Yearbook of Pediatric Endocrinology 2013
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The body of knowledge in most medical specialties is rapidly expanding, making it virtually impossible to follow all advances in clinical and basic sciences that are relevant to a given field. This is particularly true in pediatric endocrinology, at the cross-road of pediatrics, endocrinology, development and genetics. Providing abstracts of articles that report the year’s breakthrough developments in the basic sciences and evidence-based new knowledge in clinical research and clinical practice that are relevant to the field, the ‘Yearbook of Pediatric Endocrinology 2013’ keeps busy clinicians and scientists, pediatric endocrinologists, and also pediatricians and endocrinologists informed on new advances. Twelve Associate Editors and their co-authors selected from several thousand papers those that brought the most meaningful new information, summarized them and provided comments to put them into perspective. The papers are classified into those that identify new genes involved in diseases, new hormones, concepts revised or re-centered, important observations for clinical practice, large-scale clinical trials, new mechanisms, new paradigms, important review articles, new fears and new hopes. Because the Yearbook is endorsed by the European Society for Paediatric Endocrinology (ESPE), its publication is linked to the annual meeting of the ESPE. The ‘Yearbook of Pediatric Endocrinology 2013’ covers the medical and scientific literature from June 2012 through May 2013.



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Date de parution 05 septembre 2013
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EAN13 9783318025071
Langue English
Poids de l'ouvrage 1 Mo

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Yearbook of Pediatric Endocrinology 2013
Yearbook of Pediatric Endocrinology 2013
Endorsed by the European Society for Paediatric Endocrinology
Ken Ong
Ze’ev Hochberg
Associate Editors
Evangelia Charmandari
Francesco Chiarelli
Stefano Cianfarani
Mehul Dattani
Nicolas De Roux
Helmuth-Günther Dörr
Christopher Kelnar
Outi Mäkitie
Orit Pinhas-Hamiel
Michel Polak
Olle Söder
Martin Wabitsch
Sponsored by a grant from Pfizer Endocrine Care
Disclaimer. The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
© Copyright 2013 by S. Karger AG, P.O. Box, CH-4009 Basel (Switzerland)
Printed in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, Basel
ISBN 978-3-318-02506-4
e-ISBN 978-3-318-02507-1
ISSN 1662-3991

Ken Ong
Medical Research Council Epidemiology Unit
Institute of Metabolic Science, University of Cambridge, Addenbrooke's Hospital, Box 285
Cambridge CB2 0QQ, UK
Tel. +44 1223 769207, Fax+44 1223 330316, E-Mail
Ze'ev Hochberg
Faculty of Medicine
Technion-Israel Institute of Technology, POB 9602
IL-31096 Haifa, Israel
Tel. +972 50 206 1336, E-Mail
Associate Editors
Evangelia Charmandari
Division of Endocrinology, Metabolism and Diabetes
First Department of Pediatrics
University of Athens Medical School
‘Aghia Sophia’ Children's Hospital
GR-11527 Athens, Greece
Tel. +30 210 7794023, Fax+30 210 7759167, E-Mail
Francesco Chiarelli
Department of Pediatrics
University of Chieti
Via dei Vestini, 5
IT-66013 Chieti, Italy
Tel +39 0871 358015, Fax +39 0871 574831, E-Mail
Stefano Cianfarani
D.P.U.O. ‘Bambino Gesù’ Children's Hospital –
‘Tor Vergata’ University, Rome, Italy
Department of Women's and Children's Health
Karolinska Institutet, Stockholm, Sweden
P.zza Sant'Onofrio, 4
IT-00165 Rome, Italy
Tel. +39 06 6859 3074, Fax +39 06 6859 2508, E-Mail
Mehul T. Dattani
Developmental Endocrine Research Group
Clinical and Molecular Genetics Unit, Institute for Child Health
University College London
30 Guilford Street, London, WC1N 1EH, UK
Tel. +44 207 905 2657, Fax +44 207 404 6191, E-Mail
Nicolas De Roux
INSERM U 690, Laboratoire d'Hormonologie, AP-HP
Hôpital Robert Debré, 48 Boulevard Sérurier
FR-75019 Paris, France
Tel. +33 1 40 03 19 85, Fax +33 1 40 40 91 95, E-Mail
Helmuth-Günther Dörr
Pediatric Endocrinology, Department of Pediatrics
University of Erlangen, Loschgestr. 15
DE-91054 Erlangen, Germany
Tel. +49 9131 85 33732, Fax +49 9131 85 36131, E-Mail
Christopher J.H. Kelnar
Honorary Professor of Paediatric Endocrinology
University of Edinburgh
Section of Child Life and Health
Division of Reproductive and Developmental Sciences
20 Sylvan Place
Edinburgh EH9 1UW
Scotland, UK
Tel.+44 131 337 3195, Fax +44 131 536 0821, E-Mail
Outi Mäkitie
Pediatric Endocrinology and Metabolic Bone Diseases
Children's Hospital
Helsinki University Central Hospital and University of Helsinki
P.O. Box 281
FI-00029 Helsinki, Finland
Tel. +358 9 4711, Fax +358 9 471 75888, E-Mail
Orit Pinhas-Hamiel
Pediatric Endocrine and Diabetes Unit
Safra Children's Hospital
Sheba Medical Center Ramat-Gan
IL-52621 Ramat-Gan, Israel
Tel. +972 3 5305015, Fax +972 3 5305055, E-Mail
Michel Polak
Paediatric Endocrinology, Gynecology and Diabetology INSERM U845
Université Paris Descartes
Hôpital Necker-Enfants Malades
149, rue de Sèvres
FR-75015 Paris, France
Tel. +33 1 44 49 48 03/02, Fax +33 1 44 38 16 48, E-Mail
Olle Söder
Pediatric Endocrinology Unit, Q2:08
Department of Women's and Children's Health
Karolinska Institutet and University Hospital, Solna
SE-171 76 Stockholm, Sweden
Tel. +46 8 517 75124, Fax +46 8 517 75128, E-Mail
Martin Wabitsch
Division of Pediatric Endocrinology and Diabetes, and Endocrine Research Laboratory
Department of Pediatrics and Adolescent Medicine ( )
Ulm University, Eythstrasse 24
DE-89075 Ulm, Germany
Tel. +49 731 5005 7400, Fax +49 731 5005 7407, E-Mail
Table of Contents
Ze'ev Hochberg and Ken Ong
Lucie Chevrier, Brooke Tata and Nicolas de Roux
Evelien F. Gevers, Carles Gaston-Massuet and Mehul T. Dattani
Aurore Carré, Gabor Szinnai, Dulanjalee Kariyawasam, Mireille Castanet and Michel Polak
Growth and Growth Factors
Stefano Cianfarani
Growth Plate, Bone and Mineral Metabolism
Outi Mäkitie, Agnès Linglart and Terhi J. Heino
Reproductive Endocrinology
Lena Sahlin and Olle Söder
Erica L.T. van den Akker and Evangelia Charmandari
Oncology and Chronic Disease
Tilman Rohrer, Thomas M.K. Völkl and Helmuth G. Dörr
Type 1 Diabetes: Clinical and Experimental
M. Loredana Marcovecchio and Francesco Chiarelli
Obesity and Weight Regulation
Martin Wabitsch, Daniel Tews, Christian Denzer, Anja Moss, Belinda Lennerz and Pamela Fischer-Posovszky
Type 2 Diabetes, Metabolic Syndrome and Lipids
Orit Pinhas-Hamiel and Hofit Cohen
Evidence-Based Medicine in Pediatric Endocrinology
Christopher J.H. Kelnar and Roderick T. Mitchell
Editors’ Choice
Ken Ong and Ze'ev Hochberg
Science and Medicine
Ze'ev Hochberg and Ken Ong

Author Index
Subject Index
This is the 10th edition the Yearbook of Pediatric Endocrinology and while we can speak about those 10 years with some pride, we mostly think of the decade ahead. Since its initiation, the international standing of the Yearbook has increased mostly due to the team of Associate Editors, all internationally-renowned researchers. Over the next 10 years, we intend to encourage even broader coverage of emerging topics of interest, while the current balance of basic science, translational, and clinical research that is unique to the Yearbook will be continued. The rapid pace of scientific and clinical advances in pediatric endocrinology shows no sign of abating and the next 10 years should prove as exciting as the last. We look forward to playing a major role in the dissemination of new discoveries and the discussion of controversies and topical issues.
This was another inspiring year (June 2012 to May 2013) for publications. Among other reports, new treatments were described for obesity, hyperlipidemia, hypercalcemia, and Cushing's disease. New genes were identified for type 1 diabetes, obesity, Kallmann syndrome, central hypothyroidism, glucocorticoid deficiency, osteogenesis imperfecta and hypercalcemia. New mechanisms include a viral pathogenesis of obesity, the role of oxytocin in feeding behavior, the epigenetic regulation of puberty, a new component/hormone and its receptor in the renin-angiotensin system, and the evolution of maternal-fetal immune tolerance. New, or promising, clinical investigation tools were described for (cyclical) Cushing's syndrome, and antenatal and neonatal genetic diagnosis. We welcome Christopher Kelnar and his team, who have continued the high standard of the Evidence-Based Medicine chapter.
While considering our future, our history deserves to be remembered: 100 years ago, Dr. Hakaru Hashimioto recognized a new disease, now called autoimmune thyroid disorder (AITD) [ 1 ]. Even if you do not read the entire original manuscript, be impressed by the 30 pages of detailed histological descriptions of biopsies and a thorough interpretation and discussion, which was recently reviewed [ 2 ].
The support we receive from Pfizer Ltd and the professionalism of the Karger publishing team continue to underpin the progress of the Yearbook and we look forward to working with them for the next 10 years.
Ze'ev Hochberg (Haifa) Ken Ong (Cambridge)
1. Hashimoto H: Zur Kenntnis der lymphomatösen Veränderung der Schilddrüse (Struma lymphomatosa). Arch Klin Chir 1912;97:219-248.
2. Hiromatsu Y, Satoh H, Amino N: Hashimoto's thyroiditis: history and future outlook. Hormones 2013;12:12-18.
Lucie Chevrier, Brooke Tata and Nicolas de Roux
INSERM U676, Robert Debré Hospital and Paris Diderot University, Paris, France
New mechanisms Circadian rhythm and metabolism
Two different mechanisms by which circadian clock and metabolism are linked are presented in the two following papers.

Regulation of circadian behavior and metabolism by synthetic REV-ERB agonists
Solt LA, Wang Y, Banerjee S, Hughes T, Kojetin DJ, Lundasen T, Shin Y, Liu J, Cameron MD, Noel R, Yoo SH, Takahashi JS, Butler AA, Kamenecka TM, Burris TP
Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, FL, USA
Nature 2012;485:62-68
Background: The nuclear receptors REV-ERB-α and REV-ERB-β have an important role in regulation of core clock genes expression driving circadian activity and metabolism. Agonists of these receptors could be of interest to regulate circadian rhythm disorders and metabolic disorders.
Methods: The authors synthesized agonists of REV-ERB-α and REV-ERB-β and tested their effects in circadian activity and metabolic regulation.
Results: SR9011 and SR9009 are two synthetic agonists of REV-ERB-α and REV-ERB-β. In vivo, these compounds altered circadian behaviors and the expression of core clock genes in the hypothalamus of mice. The expression of metabolic genes in liver, skeletal muscle and adipose tissue was also altered, which resulted in increased energy expenditure. In obese mice, these agonists decreased body weight.
Conclusion: Agonists of REV-ERB-α and REV-ERB-β appeared to be useful pharmacological tools for the treatment of sleep disorders and metabolic diseases.
REV-ERB-α and REV-ERB-β are nuclear receptors regulating core clock genes. These receptors also play a key role in lipid synthesis. This study proposed a molecular mechanism linking circadian clock and metabolism. In fact, the authors described two new synthetic agonists of REV-ERB-α and REV-ERB-β that disrupted circadian activity and repressed clock gene expression in the hypothalamus. Interestingly, the expression of clock genes in peripheral tissues was not affected by these treatments. The circadian alteration induced by agonists of REV-ERB-α and REV-ERB-β was accompanied by changes in expression of genes involved in metabolism leading to a loss of body weight and to an increase of energy expenditure. These metabolic variations were also seen in obese mice. This study brings new insight about the development of compounds targeting REV-ERB-α and REV-ERB-β and more widely the circadian clock to treat metabolic disorders such as obesity.

Circadian rhythm of redox state regulates excitability in suprachiasmatic nucleus neurons
Wang TA, Yu YV, Govindaiah G, Ye X, Artinian L, Coleman TP, Sweedler JV, Cox CL, Gillette MU
Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL, USA
Science 2012;337:839-842
Background: Circadian rhythms are orchestrated in the suprachiasmatic nucleus (SCN) by a central circadian clock and are controlled by the circadian expression of clock genes. Redox state can regulate the expression of these genes. However, nothing was known about a potential non-transcriptional regulation of redox state on circadian rhythms.
Methods and Results: By ratiometric redox fluorometry by two-photon laser microscopy, the authors demonstrated that in the SCN, the redox state followed an endogenous circadian oscillation for which clock genes are required. Whole cell patch-clamp recording of SCN neurons showed that redox state oscillation could modify the excitability of SCN neurons through modulation of K + channels.
Conclusion: Circadian regulation of redox state by a central circadian clock modulated the excitability of SNC neurons via a non-transcriptional mechanism.
This study is interesting for two main reasons. First, it highlights that, in the suprachiasmatic nucleus (SCN), redox state shows circadian oscillations that are under the control of circadian clock genes, and brings new insight concerning circadian clock and metabolism regulation. The second reason of the interest of this study is the discovery of a novel non-transcriptional mechanism by which the circadian clock can regulate SCN neuron excitability. In fact, the redox state directly influenced the excitability of SCN neurons through modulation of K + channels, indicating that metabolic state variation could be the cause, rather than the result, of neuronal activity. Finally, it seems that at midday, circadian clock favored an oxidized state, leading to the opening of K + channels and to an increase of neuronal activity of SCN neurons. This study focused on the SCN, which is the master circadian clock. However, circadian rhythms affect the entire organism. So it is now of interest to determine if the link between circadian clock and redox state also exists in peripheral tissue.
New mechanisms GnRH and biosynthesis of neurosteroids

Gonadotropin-releasing hormone stimulates the biosynthesis of pregnenolone sulfate and dehydroepiandrosterone sulfate in the hypothalamus
Burel D, Li JH, Do-Rego JL, Wang AF, Luu-The V, Pelletier G, Tillet Y, Taragnat C, Kwon HB, Seong JY, Vaudry H
Laboratory of G-Protein-Coupled Receptors, Graduate School of Medicine, Korea University, Seoul, Republic of Korea, and Research Institute for Biomedicine (IRIB), Laboratory of Neuronal and Neuroendocrine Differentiation and Communication, Institut National de la Sante et de la Recherche Medicale U982, University of Rouen, Mont-Saint-Aignan, France or
Endocrinology 2013;154:2114-2128
Background: The enzyme hydroxysteroid sulfotransferase (HST) is involved in the biosynthesis of the sulfated neurosteroids, pregnenolone sulfate (∆ 5 PS) and dehydroepiandrosterone sulfate (DHEAS). In the frog, HST is expressed in the magnocellular nucleus and the anterior preoptic area, two hypothalamic regions that are richly innervated by GnRH1-containing fibers. However, nothing is known about the potential regulatory effect of GnRH1 on sulfated neurosteroids synthesis.
Methods and Results: Using double labeling of frog brain slices, the authors showed that GnRH1 fibers are located in close vicinity of HST-positive neurons. Three GnRH receptors were identified. RT-PCR and in situ hybridization were used to localize the three GnRH receptors and in vitro studies allowed the functional characterization of these. Pulse-chase experiments using tritiated ∆ 5 P and DHEA as steroid precursors, and 3’-phosphoadenosine 5’-phosphosulfate as a sulfonate moiety donor, showed that GnRH1 stimulated the biosynthesis of ∆ 5 PS and DHEAS in frog diencephalic explants.
Conclusion: These data suggest that some of the behavioral effects of GnRH could be mediated by the modulation of sulfated neurosteroids, known to stimulate sexual activity.
This study conducted in frog is interesting for two main points. First, the authors identified and characterized three receptors of GnRH. The wide distribution of the three GnRH receptors in the brain reflected the neurotransmitter, neuromodulator and neuroendocrine roles of GnRH. However, the precise role of each receptor was not yet known. In the pituitary, only GnRHR-1 was expressed, suggesting that this receptor is involved in the regulation of gonadotrope cells by GnRH. GnRHR-1 and GnRHR-2 were present in the testis and adrenal gland, two peripheral sites of DHEA biosynthesis. It would be interesting to determine if GnRH can modulate biosynthesis of DHEA in these steroidogenic glands. Second, this study brings new insight in neurosteroid synthesis. In fact, it underscores that in the frog, GnRH1 directly stimulates the biosynthesis of two sulfated neurosteroids, pregnenolone sulfate (∆ 5 PS) and dehydroepiandrosterone sulfate (DHEAS). It appears that the GnRH receptors GnRHR-1 and GnRHR-3 are highly expressed in the magnocellular nucleus and the anterior preoptic area, hypothalamic regions wherein GnRH1 fibers are located in close vicinity of HST-positive neurons. So, it can be thought that in the frog, stimulatory effects on GnRH1 on sexual behaviors could be mediated by sulfated neurosteroids.
New mechanism GnRH-(1-5) controls the migration of GnRH neurons

The metabolite GnRH-(1-5) inhibits the migration of immortalized GnRH neurons
Larco DO, Cho-Clark M, Mani SK, Wu TJ
Program in Molecular and Cellular Biology, Uniformed Services University of the Health Sciences, Bethesda, MD, USA
Endocrinology 2013;154:783-795
Background: GnRH is metabolized to GnRH-(1-5) by the endopeptidase EP24.15. EP24.15 is expressed along the migratory path of GnRH-expressing neurons during development. However, the potential role of GnRH-(1-5) peptide on GnRH neuron migration was unknown.
Methods: The authors used the GN11 cell line as a model of premigratory GnRH-secreting neuronal cells for in vitro studies. The role of GnRH-(1-5) on GN11 migration was determined by a wound-healing assay and RNA interference of GRP173. STAT3 pathway activation was studied by immunocytochemistry and Western immunoblotting.
Results: GnRH-(1-5) inhibited GN11 cell migration and acted via GPR173 and the inhibition of STAT3 pathway.
Conclusion: This in vitro study highlighted the potential regulatory role of GnRH-(1-5) in GnRH neuron migration during development.
Migration of GnRH neurons from the olfactory placode to the hypothalamus is a crucial step to the establishment of the hypothalamic-pituitary-gonadal axis. In fact, defect of GnRH neuron migration due to olfactory agenesis can lead to hypogonadism in Kallmann syndrome. Identification of factors involved in this migration is particularly informative to better understand the pathogenesis of Kallmann syndrome. The authors investigated the potential role of GnRH-(1-5) on the migration of GnRH neurons. The fact that EP24.15 is expressed during development along the migratory path of GnRH neurons and that migrating GnRH neurons secrete GnRH strengthens this hypothesis. In GN11 cells, an established cell line expressing GnRH, the authors showed that GnRH-(1-5) slowed migration of GN11 via the GPCR GPR173 and via the inhibition of the STAT3 pathway. GPR173 is expressed in mouse embryonic nasal tissue from E12.5, when GnRH neurons are migrating along the vomeronasal tract. This study describes an interesting autocrine effect of GnRH-(1-5) on GnRH neuron migration through GPR173 activation that needs to be tested by in vivo investigation.
New mechanisms Role of epigenetic regulations in the initiation of puberty

Changes in hypothalamic expression of the lin28/let-7 system and related microRNAs during postnatal maturation and after experimental manipulations of puberty
Sangiao-Alvarellos S, Manfredi-Lozano M, Ruiz-Pino F, Navarro VM, Sanchez-Garrido MA, Leon S, Dieguez C, Cordido F, Matagne V, Dissen GA, Ojeda SR, Pinilla L, Tena-Sempere M
Department of Cell Biology, Physiology and Immunology, Faculty of Medicine, University of Cordoba, Cordoba, Spain
Endocrinology 2013;154:942-955
Background: lin28 and lin28b are RNA-binding proteins known to inhibit the maturation of miRNAs of the let-7 family. Genome-wide association studies suggest the involvement of lin28/let-7 in the control of puberty.
Methods: The expression of lin28 and let-7 and other components of the regulatory network are studied in the rat hypothalamus during postnatal development, and in models of altered puberty.
Results: Expression of lin28, lin28b and c-Myc was high in neonatal hypothalamus, and decreased during the infantile-to-juvenile transition to reach a minimal level at puberty. let-7a, let-7b, mir-132 and mir-145 have opposite expression profiles. Perturbation of puberty onset by neonatal sex steroid treatment, by photoperiod manipulation or by subnutrition, affected the expression of the lin28/let-7 system.
Conclusions: The expression of lin28/let-7 system dramatically changed in the rat hypothalamus during the postnatal maturation and was affected in models of altered puberty, suggesting the involvement of this system in the mechanisms leading to puberty onset.
Recently, miRNAs and their regulators have been involved in the fine regulation of complex biological processes. In this study, the authors provided evidence that miRNAs are involved in the postnatal maturation of the gonadotropic axis. lin28 was highly expressed in the hypothalamus in the neonatal period and then declined at the puberty, while let-7 expression varied in the opposite direction. Delayed puberty induced by neonatal administration of sex steroids or modification of the photoperiod was associated to an alteration of the lin28/let-7 ratio in the hypothalamus. Interestingly, male and female rats did not respond equally to the modification of the photoperiod. Alterations of lin28/ let-7 expression appeared in the juvenile period in males. In females, variations of the lin28/let-7 ratio appeared in the prepubertal period. In a third model of delayed puberty induced by feeding restriction, lin28/let-7 ratio expression was not really affected. Although the precise role of lin28/let-7 system in the hypothalamus needs to be characterized, the data presented here open a new way to understand the postnatal maturation of the gonadotropic axis and how environmental factors, sex steroids or the nutrition status may interfere with this process.

Epigenetic control of female puberty
Lomniczi A, Loche A, Castellano JM, Ronnekleiv OK, Bosch M, Kaidar G, Knoll JG, Wright H, Pfeifer GP, Ojeda SR
Division of Neuroscience, Oregon National Primate Research Center, Beaverton, OR, USA
Nat Neurosci 2013; 16:281-289
Background: It is well known that not just one gene, but rather a multitude of genes control the onset of puberty. However, the genetic elements and transcriptional regulation coordinating this process for pubertal onset to occur is unknown and underexplored. Further, it does not explain how there can be dynamic plastic changes in gene networks involved at the epigenetic level for pubertal onset.
Methods: The authors injected 5-azacytidine (Aza), a DNA methyltransferase, in juvenile female Sprague-Dawley rats to inhibit DNA methylation in the hypothalamus. With this, authors measured sexual maturation and reproductive function. Next, authors used DNA methylation arrays to discover potential repressor genes at different pubertal stages. Lastly, using the Kiss-1 gene as a puberty-activating gene prototype, authors carried out various immunohistochemical and quantitative polymerase chain reaction methods to understand if these repressor genes were expressed in kisspeptin neurons and how they affected kisspeptin immunoreactivity, function, as well as GnRH pulsatility.
Results: The authors discovered that the inhibition of DNA methylation delays puberty through keeping inhibiting genes activated. These repressor genes are a part of a Polycomb Group (PcG) of proteins and particularly two genes, Eed and Cbx7 , whose mRNA levels decreased concomitantly with an increase in DNA methylation at puberty. Lastly, the authors discovered that Eed was localized at the promoter of the Kiss-1 gene and is evicted at the timing of puberty along with changes in chromatin status of Kiss1. Overexpression of Eed led to compromised reproductive capacity and reduced the number of Kiss1 -positive neurons in the ARC.
Conclusions: Hypothalamic expression of two repressive PcG genes, Cbx7 and Eed , are important for the repression of pubertal onset. Before puberty begins, expression of these genes decreases coincidently with increased DNA methylation. Eed is located at the PcG promoter and prevents the upregulation of Kiss1 mRNA before puberty, demonstrating a novel mechanism for the upregulation of Kiss1 mRNA in the ARC before puberty, involving DNA methylation and the eviction of EeD from its promoter.
The timing of puberty is associated with an increase in GnRH release. However, our focus has been mainly on the transynaptic and physiological regulation of GnRH secretion during pubertal onset. How pubertal onset in females is regulated at the epigenetic level and how this transcriptional plasticity can affect the timing of puberty is unknown. These authors investigated, for the first time, how DNA methylation contributes to the regulation of the timing of puberty in females using rats as a model. Inhibition of DNA methylation in the hypothalamus prevented puberty in female rats through the continued activation of a repressive mechanism. Using DNA methylation arrays at different pubertal stages, two genes, Cbx7 and Eed , were discovered as a part of the Polycomb Group (PcG) of transcriptional silencers whose expression decreases before the onset of puberty in association with increased DNA methylation. Using Kiss1 as a model of a puberty-activating gene to prove that the PcG complex represses the onset of puberty by targeting downstream genes responsible for the excitatory control of GnRH, the authors discovered that the PcG gene Eed is associated with the Kiss1 promoter, preventing accessibility of activating histone marks before pubertal onset. Indeed, overexpression of EeD consequently led to the reduction of immunoreactive kisspeptin neurons, decreased GnRH pulsatility, and delayed puberty. Overall, this study unveiled a novel mechanism where epigenetic silencing underlies the timing of pubertal onset. This is important in terms of human fertility and how developmental changes at the epigenetic level can have an impact on reproductive function.
New mechanism How the gonadotropic axis is inhibited at the end of the infantile period

The decline in pulsatile GnRH release, as reflected by circulating LH concentrations, during the infant-juvenile transition in the agonadal male rhesus monkey (Macaca mulatta) is associated with a reduction in kisspeptin content of KNDy neurons of the arcuate nucleus in the hypothalamus
Ramaswamy S, Dwarki K, Ali B, Gibbs RB, Plant TM
Department Obstetrics and Gynecology and Reproductive Sciences, University of Pittsburgh, Magee-Women's
Research Institute, Pittsburgh, PA, USA
Endocrinology 2013;154:1845-1853
Background: Puberty in primates is timed by two hypothalamic events — a decline in pulsatile GnRH release occurs in late infancy, which leads to a quiescent phase. Then, in late juvenile development, pulsatile GnRH release is reactivated which allows puberty to be initiated. This phase in late juvenile development is kisspeptin-dependent.
Methods: The authors conducted kisspeptin immunohistochemistry in agonadal male rhesus monkeys during early infancy when GnRH release is high with robust LH concentrations. They compared kisspeptin, neurokinin B, and GnRH immunoreactivity with male rhesus monkeys during the juvenile period when GnRH release is arrested.
Results: Although there was no difference between infant and juvenile male rhesus monkeys in terms of distribution of immunopositive kisspeptin neurons, there were more kisspeptin immunoreactive cell bodies in infant monkeys compared to juvenile monkeys. Interestingly, there was no difference in neurokinin B distribution or GnRH fiber staining intensity.
Conclusion: A reduction in the stimulatory kisspeptin tone to the GnRH neuronal network is required for the switch between the late infancy to juvenile periods in male rhesus monkeys. The reduction in kisspeptin tone guarantees the quiescent hypogonadotropic phase of the prepubertal gonad.
This article raises an interesting question, as we have always been fascinated as to how puberty is activated, but rarely have we investigated the nature of the switch that suppresses GnRH pulsatility after the minipuberty of infancy. This article addresses the question whether the loss of GnRH pulsatility in the infant primate is related to a reduction of kisspeptin in the arcuate nucleus. The findings reveal a remarkable insight into the kisspeptin-neurokinin B-dynorphin (KNDy) neuronal mechanism of GnRH pulsatility. In that first, the GnRH pulse generation that is turned off during infancy is gonad-independent and this inhibits kisspeptin neuronal activity. However, the authors discovered that the number of kisspeptin cell bodies in the ARC was unrelated to the GnRH pulse arrest, suggesting there are two separate mechanisms that regulate GnRH pulsatility and kisspeptin pre- and postpubertal onset. Furthermore, the authors discovered that, during the infant-juvenile transition, kisspeptin content in KNDy neurons significantly decreased along with its pulsatile release, leaving GnRH pulsatility arrested. However, during the juvenile-pubertal transition, kisspeptin content and pulsatility are increased, leading to an increase in GnRH. This is intriguing as these authors suggest that kisspeptin does not in fact play a regulatory role in the timing of puberty, but serves merely as an output to the GnRH pulse generator, suggesting that other mechanisms are required for these two important hypothalamic events pre- and postpubertal onset.
New gene A new gene in Kallmann syndrome

SEMA3A, a gene involved in axonal pathfinding, is mutated in patients with Kallmann syndrome
Hanchate NK, Giacobini P, Lhuillier P, Parkash J, Espy C, Fouveaut C, Leroy C, Baron S, Campagne C, Vanacker C, Collier F, Cruaud C, Meyer V, Garcia-Pinero A, Dewailly D, Cortet-Rudelli C, Gersak K, Metz C, Chabrier G, Pugeat M, Young J, Hardelin JP, Prevot V, Dode C
INSERM U837, Développement et Plasticité du Cerveau Postnatal, Centre de Recherche Jean-Pierre Aubert, Lille, France
PLoS Genet 2012;8:e1002896
Background: The genetics of the Kallmann syndrome involves various modes of transmission, including oligogenic inheritance, but mutations have been found in only 35%. Semaphorin-3A is a secreted protein acting as a guidance cue of growing axons. This protein has been shown to be critical for GnRH neuron migration in rodents. In this paper, the authors considered SEMA-3A as a candidate gene in Kallmann syndrome.
Methods: Analysis of GnRH neuron distribution in Nrp1 sema/sema mutant mice that lack a functional sema-phorin-binding domain in neuropilin-1, an obligatory co-receptor of semaphorin-3A. 386 unrelated patients were screened for SEMA-3A mutations.
Results: Nrp1 sema/sema showed abnormal development of the peripheral olfactory system and defective embryonic migration of the neuroendocrine GnRH cells to the basal forebrain, which results in increased mortality of newborn mice and reduced fertility in adults. Nonsynonymous mutations were observed in 24 patients, specifically, a frameshifting small deletion and seven missense mutations leading to low secretion or reduced signaling activity.
Conclusion: Semaphorin-3A signaling insufficiency contributes to the pathogenesis of KS and further substantiates the oligogenic pattern of inheritance in this developmental disorder.
SEMA-3A is a new mutated gene in Kallmann syndrome (KS). With 4 other genes encoding for proteins modulating the FGFR1 signaling pathway and recently described [ 1 ], more than 20 genes have now been linked to congenital hypogonadotropic hypogonadism with or without olfactory bulb agenesis. This study is interesting as it provides a new pathological mechanism to explain defects in olfactory bulb agenesis and therefore GnRH neuron migration. All SEMA-3A mutations were heterozygous in KS patients. All but one mutation were very rare. Eight mutations were shown to be loss-of-function, which confirms the model observed in Sema-3A knockout mice [ 2 ]. However, these heterozygous mutations do not fully explain the KS phenotype. The authors propose that monoallelic mutations in SEMA3A are insufficient to induce olfactory bulb agenesis, but probably act in concert with other mutations in genes regulating the semaphorin-3A signaling pathway. A similar conclusion was drawn by Young et al. [ 3 ] who also reported a SEMA-3A mutation in a family with KS.
New concern Endocrine disruptor exposure during fetal life and adult obesity

Organizational effects of perinatal exposure to bisphenol-A and diethylstilbestrol on arcuate nucleus circuitry controlling food intake and energy expenditure in male and female CD-1 mice
Mackay H, Patterson ZR, Khazall R, Patel S, Tsirlin D, Abizaid A
Department of Neuroscience, Carleton University, Ottawa, Ont., Canada
Endocrinology 2013;154:1465-1475
Background: Bisphenol-A (BPA) is a plasticizer and a known endocrine disrupter that can act as an agonist to estrogen receptors and function as an obesogen, possibly causing disrupted hypothalamic energy balance circuitry to the melanocortin system in the arcuate nucleus (ARC) of the hypothalamus.
Methods: Pregnant CD-1 female mice were subjected to environmentally relevant doses of BPA. Offspring were weaned initially onto a normal (AIN93G) diet, then as adults exposed to either a normal or high-fat diet (HFD). During adulthood, metabolic parameters, diet changes, quantification of neurons involved in the regulation of metabolism, and other parameters were measured to study the effects of early life exposure to BPA.
Results: High BPA exposure during early life (in utero and before weaning) in rodents had differential effects between adult male and female mice, as these systems are sexually differentiated. In female offspring, high BPA exposure and high-fat diet during early life led to increased weight gain, adiposity, hyperleptinemia, and masculinization of the melanocortin system, as shown by similar ER-α expression as males. However, males exposed to high BPA and high-fat diet in the same critical period exhibited impaired glucose tolerance and hyperleptinemia, but no change in body mass.
Conclusion: Early life exposure to BPA resulted in sexual dimorphic alterations in the organization and structure of the hypothalamic melanocortin pathway, leading to increased susceptibility to diet-induced diabetes, glucose intolerance and other metabolic impairments.
Bisphenol-A (BPA), a plastic monomer, has structural similarity to 17β-estradiol, acting as an agonist to the estrogen receptor (ER), and has been implicated as an endocrine disrupter and acts as an obesogen. Of particular interest and concern, BPA exposure can lead to obesity and metabolic dysfunction. The authors posed the question of how early life BPA exposure affects adult metabolic phenotype and hypothalamic energy balance. This is interesting due to the fact that the human population has ubiquitous oral exposure to BPA on a daily basis, leading to higher vulnerability for obesity, metabolic impairments, and sexual dimorphic alterations in the brain if exposed early in life. This further highlights an important point given that BPA can cross the placental barrier. Furthermore, high doses of exposure to BPA, in combination with dietary challenges, elicits a differential response in the melanocortin pathway in male versus female adults born from mothers subjected to either a high or low dose of BPA. Sexually dimorphic changes in the metabolic phenotype along with differential organization of the melanocortin pathway in the ARC were observed with early life BPA exposure. Indeed, high levels of BPA in association with a high-fat diet masculinized the female brain, predisposing to diet-induced obesity. However, with this the authors raise an important point that there is a degree of ubiquity of BPA in the environment and how to understand the mechanisms in rodent models raises controversy due to the difficulty in experimental design. However, this article overall highlights a major problem that pregnant mothers may predispose their children to diet-induced obesity through environmental estrogens such as BPA.
New hope A new pharmacological target in obesity

Obesity-driven synaptic remodeling affects endocannabinoid control of orexinergic neurons
Cristino L, Busetto G, Imperatore R, Ferrandino I, Palomba L, Silvestri C, Petrosino S, Orlando P, Bentivoglio M, Mackie K, Di Marzo V
Endocannabinoid Research Group, Institute of Cybernetics ‘Eduardo Caianiello’, and Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy
Proc Natl Acad Sci USA 2013;110:E2229-2238
Background: Alterations in energy status, chronic or acute, can change the balance between excitatory and inhibitory synaptic transmission and plasticity in the hypothalamus, which is important for the adaption of energy metabolism to new homeostatic requirements. The endocannabinoid and cannabinoid receptor type-1 (CB-1) are important modulators of excitatory and inhibitory neurotransmission, are sensitive to leptin, and their tone is altered during obesity.
Methods: To understand if the endocannabinoid system plays an important role in the readjustment of both excitatory and inhibitory inputs into the hypothalamic circuits regulating energy status, the authors used confocal and ultrastructural microscopic analyses and patch-clamp recordings in the leptin-deficient knockout mouse (ob/ob) and in mice with diet-induced obesity (DIO).
Results: As endocannabinoids act retrogradely at presynaptic sites to activate CB-1, they inhibit neurotransmitter release and fine tune synaptic transmission. In ob/ob mice and mice with DIO, the authors found that orexigenic (appetite-stimulating) neurons undergo a shift from excitatory to inhibitory control via CB-1 inputs. Further, endocannabinoid biosynthesis is increased in these mice, causing an inhibition of orexigenic neurons, leading to hyperphagia, increased body mass, as well as other metabolic problems.
Conclusion: The authors provide initial evidence that increased inhibitory control of orexigenic neurons and their CB1-mediated disinhibition were a consequence of impaired leptin signaling in the arcuate nucleus of the hypothalamus, as the metabolic dysregulation observed was reversed through leptin administration.
Endocannabinoids are important for regulation of energy status amongst other physiological functions where they act to activate retrogradely at presynaptic sites, binding to the cannabinoid receptor 1 (CB1) to inhibit neurotransmitter release and fine tune synaptic transmission. The purpose of this study was to analyze whether CB1 expressing excitatory versus inhibitory inputs to orexin-A neurons in the hypothalamus is affected during obesity and how obesity could thus affect endocannabinoid control of orexin-A neurons. Due to the sensitivity of the endocannabinoid system to leptin, the authors studied leptin-deficient (ob/ob) mice and discovered an obesity-related switch of endocannabinoid-mediated retrograde modulation of neuronal activity. With this, leptin deprivation was found to induce changes in synaptic innervation of the hypothalamus, leading to the remodeling of CB1 expressing inhibitory/excitatory fibers. This study reports a novel example in which endocannabinoid control of excitatory and inhibitory neurotransmission can be greatly changed during obesity due to leptin-dependent reorganization of glutamatergic and GABAergic synapses. The full functional implications of this mechanism need to be further investigated. In conclusion, this article provides novel insight for the development of new drugs against obesity.
New mechanism How thyroid hormones are involved in the central control of cardiovascular functions

Thyroid hormone is required for hypothalamic neurons regulating cardiovascular functions
Mittag J, Lyons DJ, Sallstrom J, Vujovic M, Dudazy-Gralla S, Warner A, Wallis K, Alkemade A, Nordstrom K, Monyer H, Broberger C, Arner A, Vennstrom B
Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
J Clin Invest 2013;123:509-516
Background: Thyroid hormone is a well-known regulator of metabolic and cardiovascular functions. Molecular mechanisms involved in the central cardiovascular control by thyroid hormone are poorly understood.
Methods and Results: Using a mouse model with a heterozygous mutation of TRα1 (Thra1 +/m mice), the authors identified an unknown population of parvalbumin (pv)-expressing neurons in the anterior hypothalamus which is dramatically decreased (70%) when thyroid hormone signaling is altered. Electrophysiological studies of these cells underscored a temperature and a thyrotropin-releasing-hor-mone sensitivity. Ablation of parvalbuminergic neurons resulted in hypertension and temperature-dependent tachycardia.
Conclusions: The authors identified a novel population of hypothalamic neurons expressing pv that required thyroid hormone signaling for proper development and that are involved in blood pressure. These findings provided a connection between hypothyroidism and cardiovascular disorders.
The central regulation of cardiovascular functions by thyroid hormones was suggested but the anatomical site was unknown. This study is particularly interesting because it leads to the identification of a population of parvalbumin (pv) neurons in the anterior hypothalamus involved in the central regulation of cardiovascular functions by thyroid hormone. In hypothyroid mice due to impaired thyroid hormone signaling, this pv cell population was reduced because of a developmental defect. Electrophysiological studies identified at least four subpopulations of parvalbuminergic cells in the anterior hypothalamus, but it was not possible to better differentiate these subpopulations. Specific ablation of these pv neurons in the anterior hypothalamus by a sophisticated method using stereotaxic injection of a novel conditionally neurotoxic adeno-associated virus into the anterior hypothalamus of pv-Cre transgenic mice indicated that pv neurons regulate blood pressure directly by the autonomic nervous system rather than an endocrine system. Even if the molecular mechanisms of central regulation of cardiovascular function by thyroid hormone need to be specified, these data may have an important clinical implication for the monitoring of the cardiovascular functions in hypothyroidism patients.
New mechanism The role of glial cells in the control of TSH expression

Coordination of hypothalamic and pituitary T 3 production regulates TSH expression
Fonseca TL, Correa-Medina M, Campos MP, Wittmann G, Werneck-de-Castro JP, Arrojo EDR, Mora-Garzon M, Ueta CB, Caicedo A, Fekete C, Gereben B, Lechan RM, Bianco AC
Division of Endocrinology, Diabetes and Metabolism, Miller School of Medicine, University of Miami, Miami, FL, USA
J Clin Invest 2013;123:1492-1500
Background: Type II deiodinase (D 2 ) activates thyroid hormone by converting thyroxine (T 4 ) to 3,5,3’-triiodothyronine (T 3 ). This allows plasma T 4 to signal a negative feedback loop that inhibits production of thyrotropin-releasing hormone (TRH) in the mediobasal hypothalamus (MBH) and thyroid-stimulating hormone (TSH) in the pituitary.
Methods: Two mouse strains with pituitary- and astrocyte-specific D 2 knockdown were developed by crossing D 2 -lox/lox mice with mice specifically expressing the Cre recombinase in thyreotrope or astrocytes. The homeostasis of the hypothalamo-pituitary thyroid was then investigated.
Results: Deletion of D 2 in thyreotropes (pit-D 2 KO) resulted in high serum T 4 , normal serum T 3 leading to euthyroidism, and a 3-fold elevation in serum TSH levels with a 40% reduction in biological activity. This decrease of the TSH bioactivity was the result of the elevated serum T 4 that increased D 2 -mediated T 3 production in the MBH, thus decreasing Trh mRNA by a negative feedback on D 2 expressed in tanycytes. D 2 deletion in astrocytes did not affect the HPT.
Conclusion: Despite near-complete loss of brain D 2 , tanycyte D 2 was preserved in astro-D 2 KO mice at levels that were sufficient to maintain both the T 4 -dependent negative feedback loop and thyroid economy. Taken together, these data demonstrated that the hypothalamic-thyroid axis is wired to maintain normal plasma T 3 levels, which is achieved through coordination of T 4 -to-T 3 conversion between thyrotrophs and tanycytes.
This paper describes an interesting new loop of regulation of the thyroid axis. This loop involves interaction between neurons and glial cells in the hypothalamus. A functional interaction between tanycytes and TRH bioactivity was already known. Indeed tanycytes have the capacity to cleave proTRH in active TRH peptides. Here, the authors showed the link between D 2 expression in tanycytes and Trh expression in neurons. This results from the regulation of T 3 production by D 2 expressed in tanycytes. The second main result showed that TSH bioactivity was decreased in mice with a conditional deletion of D 2 in thyrotropes. This decrease of TSH bioactivity is due to an alteration in TSH glycosylation resulting from the low expression of Trh in the hypothalamus. This increase in TSH levels with reduced TSH bioactivity has a neutral effect on thyroidal activity. Further work will define whether this role of tanycytic D 2 in the feedback regulation mechanism on the hypothalamo-pituitary regulation of the thyroidotropic axis in mouse is also relevant in humans.
New mechanism Role of AGRP hypothalamic networks in hunger

Deconstruction of a neural circuit for hunger
Atasoy D, Betley JN, Su HH, Sternson SM
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA
Nature 2012;488:172-177
Background: Hunger is a complex behavioral state that elicits intense food seeking and consumption. Activation of starvation-sensitive AGRP neurons evokes voracious eating even in well-fed mice. Inversely, ablation of AGRP neurons results in aphagia. However, synaptic interactions of AGRP neurons with other cell populations need to be elucidated.
Methods: The authors used optogenetic and pharmacogenetic techniques to map neuronal interactions with AGRP neurons.
Results: An inhibitory circuit with paraventricular hypothalamus (PVH) neurons substantially accounted for acute AGRP neuron-evoked eating. Within the PVH, the authors found that AGRP neurons target and inhibit oxytocin neurons in the forebrain, a small population that is selectively lost in Prader-Willi syndrome, a condition involving insatiable hunger.
Conclusion: By developing strategies for evaluating molecularly defined circuits, the authors show that AGRP neuron suppression of oxytocin neurons is critical for evoked feeding. These experiments reveal a new neural circuit that regulates hunger state and pathways associated with overeating disorders.
This paper reports a highly detailed analysis of three neuronal networks involving AGRP neurons. These results were mainly based on the optogenetic approach, which is a very powerful technique to characterize the neuronal network. One of these networks, involving AGRP neurons in feeding behavior, relies on the inhibition of POMC neurons which are more involved in longer-term rather than acute feeding responses. A second network inhibits paraventricular hypothalamic neurons through synaptic contacts with AGRP neurons. This second network controls acute AGRP neuronevoked feeding. The fact that AGRP neurons exert an inhibitory effect on oxytocin neurons located in the paraventricular hypothalamus represents a new pathway in the control of hunger. This result confirms the known function of the central oxytocin in controlling feeding. In addition to the description of this new pathway, the authors suggest that the insatiable hunger in patients with Prader-Willi syndrome is due to the loss of oxytocin neurons reported in this syndrome. Altogether, the characterization of this new circuit that regulates hunger suggests new therapeutic targets in feeding disorders.
New mechanism How ghrelin bypasses the blood-brain barrier to modulate hypothalamic neuron plasticity

Rapid sensing of circulating ghrelin by hypothalamic appetite-modifying neurons
Schaeffer M, Langlet F, Lafont C, Molino F, Hodson DJ, Roux T, Lamarque L, Verdie P, Bourrier E, Dehouck B, Baneres JL, Martinez J, Mery PF, Marie J, Trinquet E, Fehrentz JA, Prevot V, Mollard P
Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5203, Institut de Génomique Fonctionnelle, Montpellier, France
Proc Natl Acad Sci USA 2013;110:1512-1517
Background: The neurons of the arcuate nucleus (ARH) are critical in the sense that they must be able to rapidly sense and integrate peripheral signals to maintain metabolic homeostasis.
Methods: The authors developed a novel technique of in vivo multiphoton microscopy along with fluorescently labeled ghrelin to demonstrate in real-time the extravasation of ghrelin from the periphery into the ARH in order to understand the mechanism how blood-borne molecules can rapidly access their target neurons by bypassing the blood-brain barrier.
Results: The authors discovered that ghrelin rapidly binds neuropeptide-Y and pro-opiomelanocortin (POMC) neurons in the near vicinity of the fenestrated capillaries of the median eminence (ME), whose vessels are connected to the ventromedial ARC (vmARH). With this, the authors observed plasticity in the system, in that ghrelin-positive neurons varied with feeding status.
Conclusions: The hypothalamus, in particular the ARH, is able to not only directly sense peripheral signals, but also modify energy status in a nutrient-dependent plastic manner.
Satiety signals fluctuate on an ultradian rhythm where peripheral food intake signals must be rapidly sensed centrally for homeostatic regulation of feeding responses. However, the mechanisms by which peripheral blood-borne molecules circumvent the blood-brain barrier and reach the arcuate nucleus (ARH) to enable rapid homeostatic regulation of energy status is currently underexplored. The authors developed a novel tool to study this mechanism using in vivo multiphoton microscopy in real time with tractable bioactive ghrelin. They discovered that circulating fluorescently labeled ghrelin can extravasate through the fenestrated capillaries of the median eminence (ME), whose vessels project to the ARH, to rapidly bind to their target sites and centrally regulate metabolic signals in a plastic manner. This article is novel and important in the aspect of not only the development of a new technology to study how peripheral metabolic molecules can be sensed centrally in a rapid manner, but also how this process for metabolism is plastic, which is required for the dynamic sensing of the hypothalamus to external feeding patterns to be able to adapt in a plastic fashion. This is of utmost importance when considering clinical pathologies such as obesity and diabetes. In all, this study opens new doors for both the study of how peripheral metabolic signals can be sensed rapidly and in a plastic manner via the ME, and also unveils potential access routes for other molecules that integrate between the periphery and the brain.
1. Miraoui H, Dwyer AA, Sykiotis GP, Plummer L, Chung W, Feng B, et al: Mutations in FGF17, IL17RD, DUSP6, SPRY4, and FLRT3 are identified in individuals with congenital hypogonadotropic hypogonadism. Am J Hum Genet 2013;92:725-743.
2. Cariboni A, Davidson K, Rakic S, Maggi R, Parnavelas JG, Ruhrberg C: Defective gonadotropin-releasing hormone neuron migration in mice lacking SEMA3A signalling through NRP1 and NRP2: implications for the aetiology of hypogonadotropic hypogonadism. Hum Mol Genet 2011;20:336-344.
3. Young J, Metay C, Bouligand J, Tou B, Francou B, Maione L, et al: SEMA3A deletion in a family with Kallmann syndrome validates the role of semaphorin 3A in human puberty and olfactory system development. Hum Reprod 2012;27:1460-1465.
Evelien F. Gevers a , b , Carles Gaston-Massuet b and Mehul T. Dattani c , d
a Department of Paediatric Endocrinology, The Royal London Hospital, Barts Health Trust, London, b Department of Endocrinology, William Harvey Research Institute, Queen Mary University London, London, c Developmental Endocrinology Research Group, Clinical and Molecular Genetics Unit, Institute for Child Health, London, and d Department of Endocrinology, Great Ormond Street Hospital, London, UK
This was yet another year with some breakthrough papers in the field of pituitary research. Mutations in the X-linked gene IGSF1 were found to underlie central hypothyroidism with macro-orchidism, although the function of the gene is still unknown. Another paper showed how Pax7 controls melanotrope lineage differentiation through chromatin modeling, allowing Tpit to bind regulatory regions for melanotrope differentiation. For the first time, inactivating ACTH mutations were found as a cause for adrenal insufficiency. Following on from the impressive work on the role of oxytocin in the development of the neurovascular interface of the pituitary is work on FGF3 and FGF10 showing that these growth factors are involved in both axonal guidance and hypophyseal vascularization. There were several clinical papers on Cushing's syndrome – one showing the outcome of surgery in Cushing's disease in an impressive number of patients (n = 200) from a single center, another on the recovery of the adrenal axis after treatment for Cushing disease, and a state-of-the-art review on the diagnosis and management of Cushing's syndrome. Work on SOX2 always continues and this year there are papers on SOX2 and pituitary stem cells, SOX2 and pituitary development and SOX2 and embryonic stem cell differentiation. Lots to choose from!
Mechanism of the year

The selector gene Pax7 dictates alternate pituitary cell fates through its pioneer action on chromatin remodeling
Budry L, Balsalobre A, Gauthier Y, Khetchoumian K, L'Honore A, Vallette S, Brue T, Figarella-Branger D, Meij B, Drouin J
Laboratoire de Génétique Moléculaire, Institut de Recherches Cliniques de Montréal (IRCM), Montréal, Qué., Canada
Genes Dev 2012;26:2299-2310
Background: During organogenesis, selector genes work by defining positional identity so that terminal differentiation within domains or tissue compartments can occur. One example of selector genes in the central nervous system is the Pax family of transcription factors. Thus in the neural tube, Pax3 and Pax6 are expressed dorsally and confer dorsal identity so that dorsal neural tube cell types can terminally differentiate. The molecular mechanisms by which these selector genes control the transcriptional programs remain elusive to date. The pituitary gland is an ideal organ to study the mechanism of terminal differentiation as it contains six secretory cell types. A number of transcription factors important for terminal differentiation of the adult pituitary gland have been identified, and amongst these is Tpit, a T-box-containing transcription factor which is critical for POMC (propiomelanocortin) transcription. POMC is expressed in two lineages: the corticotropes in the anterior pituitary (AP) gland and the melanotropes in the intermediate lobe (IL). Inactivation of Tpit leads to absence of POMC-expressing cells of both the melanotrope and the corticotrope lineages and TPIT mutations in humans cause isolated ACTH deficiency in children. How Tpit acts to generate two lineages of distinct identity, namely the melanotropes and corticotropes, remains unknown. This article identifies, for the first time, the transcription factor Pax7 as a selector gene switch that promotes melanotrope-specific genes at the expense of corticotropes, hence providing a novel mechanism by which selector genes act during terminal differentiation.
Methods: Expression profiling of differentially expressed genes between the IL and AP. Expression profiling to identify the melanotrope and corticotrope-specific cell lineages. Analyses of transgenic mice mutant for Pax7 and Tpit to assess the differentiation potential of these transcription factors. ChIP-Seq and FAIRE sequencing to identify chromatin remodeling and transgenic Pax7 gain of function using the Pitx1 promoter.
Results: Pax7 was differentially expressed in the intermediate lobe of the pituitary gland preceding Tpit and POMC expression. The restricted expression pattern of Pax7 in the IL suggests a role for this factor in melanotrope differentiation. Indeed, Pax7 null embryos exhibited decreased POMC and PC2 expression specifically in the melanotropes of the IL, whilst expression of the Tpit lineage marker remained unchanged. Interestingly, there was a change from melanotrope to corticotrope fate in the absence of Pax7, as seen by the change of the transcriptional signature of IL-expressing genes in the Pax7 -/- pituitaries. Overexpression of Pax7 under the control of the Pitx1 promoter led to exhaustion of the progenitor cell pool by promoting their differentiation. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and FAIRE (formaldehyde-assisted isolation of regulatory elements) sequencing revealed regions of accessible DNA; interestingly, Pax7-expressing cells exhibited active chromatin marks, consistent with a role for Pax7 in chromatin remodeling that would allow Tpit binding to certain promoter regions that promote melanotrope fate. This chromatin remodeling effect makes Pax7 a pioneer transcription factor with a role in promoting binding of Tpit to certain enhancers of the melanotrope lineage differentiation.
Conclusion: Taken together, these findings clearly identify a novel mechanism by which Tpit , through the selector gene Pax7 , acts to generate two distinct lineages within the pituitary gland – the melanotropes and corticotropes. Pax7 acts as a selector gene that functions as a genetic switch to promote melanotrope lineage. This is accomplished through its pioneering activity by changing chromatin conformation at specific genomic loci, so that Tpit can bind and activate melanotrope lineage-specific genes.
In this excellent piece of work from Jacques Drouin's laboratory, a new molecular mechanism that controls the specification of two distinct cell lineages, namely melanotropes and corticotropes, by the same transcription factor Tpit, has been established. Melanotropes and corticotropes both express POMC, but melanotropes are located in the intermediate pituitary lobe and cleave POMC to α-MSH, and corticotropes are located in the anterior lobe. In a series of elegant studies, Pax7 is shown to be a selector gene that confers cell identity towards the melanotrope lineage. Hence, the question of how a single transcription factor Tpit governs the differentiation of the two distinct POMC lineages has been answered. In this process, Pax7 exerts pioneering activity by chromatin remodeling specific sites so that Tpit can transcriptionally activate the melanotrope gene expression program. In the absence of Pax7 , melanotrope differentiation fails to occur despite Tpit expression being unchanged. The mechanism reported in this paper provides us with a novel molecular mechanism by which the pituitary is built. As is the case with all good papers, it opens doors to some important questions: Is there a selector gene for the corticotrope lineages too? What is the specific role of Pax7 in some of the reported Pax7-positive Cushing adenomas?
New gene – but what does it do?

Loss-of-function mutations in IGSF1 cause an X-linked syndrome of central hypothyroidism and testicular enlargement
Sun Y, Bak B, Schoenmakers N, van Trotsenburg AS, Oostdijk W, Voshol P, Cambridge E, White JK, Le Tissier P, Gharavy SN, Martinez-Barbera JP, Stokvis-Brantsma WH, Vulsma T, Kempers MJ, Persani L, Campi I, Bonomi M, Beck-Peccoz P, Zhu H, Davis TM, Hokken-Koelega AC, Del Blanco DG, Rangasami JJ, Ruivenkamp CA, Laros JF, Kriek M, Kant SG, Bosch CA, Biermasz NR, Appelman-Dijkstra NM, Corssmit EP, Hovens GC, Pereira AM, den Dunnen JT, Wade MG, Breuning MH, Hennekam RC, Chatterjee K, Dattani MT, Wit JM, Bernard DJ
Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands
Nat Genet 2012;44:1375-1381
Background: Congenital central hypothyroidism (CH) occurs either in isolation or in conjunction with other pituitary hormone deficits. Isolated CH is rare and has been described in association with mutations in thyrotropin-releasing hormone receptor (TRHR) or TSH β .
Methods: Exome and candidate gene sequencing was used to identify the molecular basis of central hypothyroidism in patients in whom sequencing of known causes of CH had failed to establish the molecular basis.
Results: The authors identified 8 distinct mutations and 2 deletions in IGSF1 in males from 11 unrelated families with central hypothyroidism, testicular enlargement and variably low prolactin concentrations. IGSF1 encodes a membrane glycoprotein that is highly expressed in the anterior pituitary gland. Wild-type and mutant forms of IGSF1 were expressed in heterologous cells, and all of the mutants exhibited immature patterns of glycosylation relative to wild-type, suggestive of their retention in the endoplasmic reticulum. Immunofluorescence and cell-surface biotinylation analyses confirmed that the mutant proteins were either blocked or significantly impaired in their trafficking to the plasma membrane. Igsf1 -deficient (Igsf1∆ ex1 ) male mice showed diminished pituitary and serum thyroid-stimulating hormone (TSH) concentrations, reduced pituitary thyrotropin-releasing hormone (TRH) receptor expression, decreased triiodothyronine concentrations and increased body mass.
Conclusions: The authors describe a new X-linked disorder, in which loss-of-function mutations in IGSF1 cause central hypothyroidism, possibly secondary to an associated impairment in pituitary TRH signaling.
This paper reports a novel syndrome characterized by central hypothyroidism and macro-orchidism. Whole exome sequencing performed independently by two groups led to the identification of loss of function mutations in IGSF1 , a membrane glycoprotein that is expressed in the pituitary. The function of the gene remains unknown, however a murine model demonstrated a subtle form of hypothyroidism consistent with the human phenotype. Additionally, given the suboptimal FT 4 and TSH responses to TRH, and the reduced expression of Trhr in mutant mice, it is likely that IGSF1 in some way is implicated in TRH signaling. Additionally, Igsf1 is expressed in human and murine testis, but enlargement of the testes was not identified in the mutant mice. This might reflect a species difference. In humans, 5 of 20 female carriers were affected with mild central hypothyroidism. Three of the patients manifested GH deficiency that later reversed, and 18 of 26 cases were prolactindeficient, in keeping with the detection of IGSF1 protein in mouse lactotropes, somatotropes and thyrotropes, but not in gonadotropes. Importantly, identification of the molecular basis of central hypothyroidism in the probands led to the identification of further members of the family in whom central hypothyroidism had previously been undetected, with subsequent commencement of thyroxine.
Further studies will shed light on this unusual phenotype, in particular the GH deficiency. (Is it truly present, is it reversible, is the GH secretory pattern disturbed?), and the macro-orchidism. Additionally, it is likely that the murine mutant used in this study, in which exon 1 was deleted from the Igsf1 gene, may act as a hypomorph as opposed to a complete loss of function. Hence, conditional transgenesis with a complete knock-out of the gene leading to complete loss of function might shed more light on the role of this gene in mouse and human.
New mechanism

Direct and indirect roles of Fgf3 and Fgf10 in innervation and vascularization of the vertebrate hypothalamic neurohypophysis
Liu F, Pogoda HM, Pearson CA, Ohyama K, Lohr H, Hammerschmidt M, Placzek M
MRC Centre for Developmental and Biomedical Genetics and Department of Biomedical Science, University of Sheffield, Sheffield, UK
Development 2013;140:1111-1122
Background: Homeostasis of the adult organism is accomplished by secretion of hormones into the bloodstream by the hypothalamic-pituitary (HP) axis. In this process neurohormones and neurotransmitters are released from axonal terminals into the vascular network that in turn targets the anterior pituitary gland and peripheral organs. To achieve this, the hypothalamo-neurohypophysis neurons (H-NH) have to establish congruent connections with the blood vessels. This process is genetically regulated and starts during embryogenesis with the innervation of the prospective hypothalamic area, where parvocellular and magnocellular neurons project their axons that will control the secretion of hormones of the anterior pituitary gland. The mechanism that controls the innervation and the vascularization of the neurohypophysis is poorly understood. In this article, 2 members of the fibroblast growth factor family of secreted proteins, Fgf3 and Fgf10, are shown to exert an axonal chemoattractive role of H-NH neurons as well as to promote NH vascularization by promoting endothelial growth.
Methods: To understand the role of Fgf3 and Fgf10 during hypothalamic development, the authors used in situ hybridization, complemented with ex vivo embryonic explant culture of the prospective hypothalamic area. Fgf3/Fgf10 blocking antibodies and chemical compounds were used to inhibit Fgf signaling. The authors studied the innervation and vascularization of transgenic mutant Fgf3 zebrafish, with temporal and conditional ablation of Fgf3 signaling using a dominant negative Gal4-UAS transgenic fish line.
Results: Fgf3 and Fgf10 were expressed during embryogenesis in the prospective hypothalamic regions where the H-NH neurons send their axonal projections. This expression pattern is at its peak when axons invade the NH, suggesting a role for these molecules as axonal chemoattractants. In order to further explore this possibility, ex vivo chick explants of prospective NH explant were co-cultured at a distance from the axonal source. This resulted in attraction of axonal projections towards the explant, thus indicating that naive NH secretes chemoattractive molecules. This chemoattraction is mediated through Fgf signaling, as inhibition of Fgf by SU5402, a chemical inhibitor, stopped axonal projections. Moreover, NH explants treated with either Fgf3 or Fgf10 blocking antibodies resulted in abrogation of axonal extension, indicating that innervation of the NH is mediated by Fgf3/Fgf10. However, treatment with high doses of Fgf10 halted axonal projections indicating that, at lower doses, Fgf10 functions as a chemoattracting agent whilst at higher doses it stops axonal projections. Beads soaked with recombinant Fgf10 and Fgf3 elicited the same effect, further supporting a role for these molecules in NH innervation. Interestingly, the prospective NH can elicit endothelial capillary vessel formation in vitro when co-cultured with chorioallantoic membrane. Only explant from early stages can elicit this function, which is abrogated by blocking of Fgf3 and Fgf10 antibodies, indicating that prospective HP can promote vascularization of NH in a timerestricted fashion. In order to assess the role of these molecules in vivo, mutant transgenic fish that lack Fgf3 showed absence of NH innervation accompanied by vascular abnormalities, further demonstrating that Fgf3 is required for innervation of the NH. Moreover, using a cell-specific blockade of Fgf signaling by expressing a dominant negative form of Fgf3 in endothelial cells resulted in vascularization defects, further demonstrating the in vivo role of Fgf3 in the wiring and vascularization of the NH.
Conclusions: Fgf3 and Fgf10 control NH innervation at early stages by acting as a chemoattractant for H-NH axons. Simultaneously they promote hypophyseal vascularization at early stages by affecting growth of endothelial cells.
Little is known about the molecular mechanism governing the formation of the posterior pituitary and hypothalamus. Axons from both the parvocellular and magnocelluar neurons have to establish congruent connections with the blood vessels for the proper secretion of hormones into the circulation. In this elegant study, Marisa Plackzec and co-workers identify for the first time a dual novel role for Fgf3/Fgf10 in innervation and vascularization of the prospective hypothalamic region, already at early developmental stages. Using both in vitro assays and chick explant assays, they were able to demonstrate that the prospective hypothalamic region secretes Fgf3 and Fgf10 that act as chemoattractive cues to neurohypothalamic neurons. Moreover, Fgf3 in both chick and zebrafish acts on endothelial cells to initiate vasculogenesis of the posterior pituitary gland.
Last year, we discussed the paper by Gutnick et al. [ 1 ], which reported that oxytocin functions as a blood vessel attractant to establish the neurovascular connection of the posterior pituitary. Two new factors, namely Fgf3 and Fgf10, now emerge as main contributors to both wiring and vascularization of the hypothalamus.
Old genes – new mutations

Bioinactive ACTH causing glucocorticoid deficiency
Samuels ME, Gallo-Payet N, Pinard S, Hasselmann C, Magne F, Patry L, Chouinard L, Schwartzentruber J, Rene P, Sawyer N, Bouvier M, Djemli A, Delvin E, Huot C, Eugene D, Deal CL, Van Vliet G, Majewski J, Deladoey J
Endocrinology Service and Research Center, Centre Hospitalier Universitaire Sainte-Justine, Department of Pediatrics, University of Montreal, Montreal, Que., Canada
J Clin Endocrinol Metab 2013;98:736-742
Background: Severe cortisol deficiency associated with elevated concentrations of ACTH is usually due to primary adrenal failure due to a number of causes including developmental disorders, autoimmune disease or infection. Other causes include familial glucocorticoid deficiency (FGD), which can be due to mutations in the genes MC2R, MRAP, MCM4 , or NNT.
Methods: The authors aimed to identify the molecular basis of the adrenal insufficiency in a 4-year-old girl and a 4-month-old boy who presented with hypoglycemia, normal electrolytes, low cortisol, and high ACTH. Both patients manifested red hair and obesity. A diagnosis of primary adrenal insufficiency was made and initial treatment was commenced with glucocorticoids and mineralocorticoids. The genes known to cause FGD were normal. Following the identification of mutations in the gene encoding POMC using whole exome sequencing, wild-type and mutant ACTH and α-MSH peptides were studied in functional assays of receptor binding and cAMP production. Methods included peptide synthesis, ACTH immunoradiometric assay, hormone binding, and activation assays in cells expressing melanocortin receptors.
Results: Exome sequencing identified compound heterozygosity for POMC mutations in the girl: one previously described null allele and one novel p.R8C mutation in the sequence encoding ACTH and α-MSH. The boy was homozygous for the p.R8C mutation. ACTH-R8C was immunoreactive, but failed to bind and activate cAMP production in melanocortin-2 receptor (MC2R)-expressing cells, and α-MSH-R8C failed to bind and stimulate cAMP production in MC1R- and MC4R-expressing cells.
Conclusions: This is the first report of glucocorticoid deficiency due to the secretion of an ACTH molecule that lacks biological bioactivity but conserves immunoreactivity, i.e. a bioinactive form of ACTH. POMC mutations should thus be considered in patients presenting with apparent ACTH resistance. The findings underline the limitations of immunoassay-based diagnostics and demonstrate the value of genetic analysis. This study also emphasizes the importance of establishing the molecular etiology of the adrenal insufficiency in these 2 individuals, which then allowed cessation of unnecessary mineralocorticoid therapy. Finally, discovery of this mutation indicates that in humans, the amino acid sequence His 6 Phe 7 Arg 8 Trp 9 is important not only for cAMP activation but also for ACTH binding to MC2R.
The authors show that mutations in POMC can be associated with a phenotype characterized by low cortisol concentrations and elevated ACTH concentrations. To date, mutations in POMC have been identified in 10 patients, all of whom have low or undetectable plasma ACTH concentrations. It is now clear that the phenotypic spectrum can be expanded to include patients with a high ACTH concentration and preservation of mineralocorticoid secretion. Hence the phenotype is similar to that of familial glucocorticoid deficiency, except that, in this study, both patients had red hair and obesity, features which are indicative of POMC mutations. The p.R145C missense POMC mutation was identified in both probands, and was included in a sequence common to ACTH and α-MSH. The mutation led to a reduction in both cAMP production and binding affinity to MC2R (ACTH) and MC1R and MC4R (ACTH, α-MSH), showing that the amino acid sequence HFRW is essential for ACTH and α-MSH binding to their respective receptors and for cAMP generation as well; in contrast, previous animal studies had suggested that binding of ACTH was dependent on the KKRRP amino acid sequence only [ 2 ]. The study shows the importance of keeping an open mind when patients present with unusual phenotypes that are not consistent with known genetic mutations. Ideally, a combination of clinical, biochemical, and genetic data are necessary to understand such unusual clinical syndromes.

Variations in PROKR2, but not PROK2, are associated with hypopituitarism and septo-optic dysplasia
McCabe MJ, Gaston-Massuet C, Gregory LC, Alatzoglou KS, Tziaferi V, Sbai O, Rondard P, Masumoto KH, Nagano M, Shigeyoshi Y, Pfeifer M, Hulse T, Buchanan CR, Pitteloud N, Martinez-Barbera JP, Dattani MT
Developmental Endocrinology Research Group, Clinical and Molecular Genetics Unit, University College London (UCL)-Institute of Child Health, London, UK
J Clin Endocrinol Metab 2013;98:E547-557
Background: Loss-of-function mutations in PROK2 and PROKR2 have been implicated in Kallmann syndrome (KS), characterized by hypogonadotropic hypogonadism and anosmia. Recent data suggest overlapping phenotypes/genotypes between KS and congenital hypopituitarism (CH), including septooptic dysplasia (SOD). Mutations in FGF8 and its receptor FGFR1 have recently been implicated in the etiology of defects of midline brain development as well as KS/isolated hypogonadotropic hypogonadism (IHH), as have PROKR2 variants.
Methods: To further characterize the role of PROKR2 in hypothalamo-pituitary development, the authors screened a cohort of patients with complex forms of CH (n = 422) for mutations in PROK2 and PROKR2 , using direct sequencing. Subsequently, a number of functional studies were performed to investigate the cell surface expression of PROKR2. Prokr2 -/- null mutant mice were analyzed using immunochemistry against a number of pituitary terminal differentiation markers.
Results: No mutations were identified in PROK2. Five PROKR2 variants were identified in 11 patients with SOD/CH: the novel p.G371R and previously reported p.A51T, p.R85L, p.L173R, and p.R268C. The latter three mutations have previously been associated with impaired function of the protein. Functional studies of the p.G371R substitution revealed no compromise in function compared to the wild-type protein. Surprisingly, a patient with SOD was heterozygous for the p. L173R variant, whereas his phenotypically unaffected mother was homozygous for the variant. Analysis of Prokr2 -/- mice revealed predominantly normal hypothalamo-pituitary development and terminal cell differentiation, with the exception of reduced LH; this was inconsistent with patient phenotypes and more analogous to the healthy mother. Importantly, the mother did not have KS, unlike the Prokr2 -/- mice.
Conclusions: The role of PROKR2 in the etiology of CH, SOD, and KS is unclear, as demonstrated by the lack of a phenotype-genotype correlation, and the association of clinical phenotypes with loss-of-function variants in both heterozygosity or homozygosity. The authors report a phenotypically normal parent, homozygous for the functionally deleterious p.L173R substitution. These data suggest that the variants identified here are unlikely to be implicated in isolation in these disorders; other genetic or environmental modifiers may also impact on the etiology. Given the phenotypic variability, genetic counseling may presently be inappropriate.

PROKR2 variants in multiple hypopituitarism with pituitary stalk interruption
Reynaud R, Jayakody SA, Monnier C, Saveanu A, Bouligand J, Guedj AM, Simonin G, Lecomte P, Barlier A, Rondard P, Martinez-Barbera JP, Guiochon-Mantel A, Brue T
Centre de Recherche en Neurobiologie et Neurophysiologie de Marseille (CRN2M), Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7286, Aix-Marseille University, France
J Clin Endocrinol Metab 2012;97:E1068-1073
Background: Pituitary stalk interruption syndrome (PSIS) represents a frequent feature of congenital hypopituitarism, but only rarely has a genetic cause been identified. To date, mutations in HESX1, LHX4, SOX3 and OTX2 have been implicated in children with PSIS. The authors hypothesized that an ectopic posterior pituitary may be a consequence of defective neuronal axon projections along the pituitary stalk or defective angiogenesis of the hypophyseal portal circulation, due to defects in the prokineticin 2 pathway.
Methods: PROK2 and PROKR2 and all genes previously known to be involved in hypopituitarism with pituitary stalk interruption (LHX4, HESX1, OTX2 , and SOX3) were screened for mutations in 72 index cases with pituitary stalk interruption syndrome from the GENHYPOPIT database. In vitro studies were performed to assess the functional consequences of allelic variants, using intracellular calcium mobilization assays and cell surface expression studies for PROKR2 and DNA binding studies and transcriptional repression assays for HESX1.
Results: The authors identified two heterozygous PROKR2 mutations (p.Leu173Arg and p.Arg85His), previously reported in Kallmann syndrome or isolated hypogonadotrophic hypogonadism, and a novel PROKR2 variant (p.Ala51Thr). The p.Ala51Thr variant did not have a deleterious effect on the protein, whereas the p.Leu173Arg substitution led to impaired cell surface targeting of the receptor and the p.Arg85His substitution affected G protein coupling of the receptor. Three allelic variants of HESX1 were identified: the heterozygous p.Phe156Ser and the homozygous p.Arg109X mutations were functionally deleterious, whereas p.Ser67Thr was found as a rare allelic variant in association with the p.Arg85His PROKR2 mutation in the same patient.
Conclusions: The authors reported PROKR2 variants in patients with congenital hypopituitarism with pituitary stalk interruption, suggesting a potential role of the prokineticin pathway in pituitary development.
The above pair of papers shed interesting and novel insights into the etiology of congenital hypopituitarism and the role of PROKR2 therein. Given the role of other genes implicated in the etiology of Kallmann syndrome (KS) in complex midline abnormalities of the brain and hypothalamo-pituitary region [ 3 , 4 ], it made sense to look for mutations in PROKR2 and its ligand PROK2. Surprisingly, both groups reported a relatively high incidence of genetic variants in PROKR2 (2% of a large complex cohort of 422 patients with hypopituitarism/SOD in the study by McCabe et al., 2.8% in a study of 72 patients with pituitary stalk interruption in the paper by Reynaud et al.). Importantly, many of the variants had been shown previously to be implicated in KS, and to lead to a loss or impaired function. As observed previously, mutations may be homozygous or heterozygous, and variably penetrant. In the article by Reynaud et al., the unaffected mother and sister of case A.II.1 both harbored the same genetic change, namely the functionally significant p. Leu173Arg, as did the asymptomatic father of cases B.II.1 and B.II.3 who harbored the same change as his 2 daughters (p.Ala51Thr, possibly a polymorphic variant). Most surprisingly, the unaffected mother of patient VI in the article by McCabe et al. was homozygous for the p.Leu173Arg variant, yet had no evidence of KS, IHH or hypothalamic amenorrhea. However, patient VI had evidence of SOD. These data suggest possible digenic/oligogenic inheritance. Recently, Avbelj Stefania et al. [ 5 ] reported that the PROKR2 L173R represents a founder mutation whose age is estimated at approximately 9,000 years. Inheritance of PROKR2 L173R-associated GnRH deficiency is complex with highly variable penetrance among carriers, influenced by additional mutations in the other PROKR2 allele (recessive inheritance) or another gene (digenicity). Indeed, Reynaud et al. reported that a further child born to consanguineous parents (case C.II.1) harbored the previously described p. Arg85His variant in PROKR2 in association with the p.Ser67Thr variant in HESX1. The authors suggest an oligogenic inheritance, however they found no deleterious effect of the HESX1 variant, and so this conclusion must remain speculative.
McCabe et al. also examined the hypothalamus and pituitary of Prokr2 -/- null mutant mice, and reported no reduction in expression of terminal differentiation markers apart from LH, as described previously [ 6 ]. The pituitary glands were small, in keeping with a reduction in the overall size of the animals. Together with the data from human patients with PROKR2 mutations, these findings suggest that mProkr2 is dispensable for proper formation of the hypothalamic-pituitary axis. This is in keeping with data in humans that also suggest that PROKR2 mutations are not, in isolation, causative of KS, IHH, hypothalamic amenorrhea and SOD/CPHD. They are likely, however, to contribute to the various phenotypes in combination with other genetic mutations and/or environmental factors, a rich area for future research.
New hope

The adult pituitary shows stem/progenitor cell activation in response to injury and is capable of regeneration
Fu Q, Gremeaux L, Luque RM, Liekens D, Chen J, Buch T, Waisman A, Kineman R, Vankelecom H
Laboratory of Tissue Plasticity, Research Unit of Embryo and Stem Cells, Department of Development and Regeneration, University of Leuven (Katholieke Universiteit Leuven), Campus Gasthuisberg (O&N1), Leuven, Belgium
Endocrinology 2012;153:3224-3235
Background: In this paper, a transgenic mouse model was constructed to conditionally ablate somatotropes to investigate the regenerative capacity of the adult pituitary and the response of its stem/progenitor cell compartment to damage.
Methods: GHCre/iDTR mice express diphtheria toxin (DT) receptor only after transcriptional activation by GH promoter-driven Cre recombinase. Treatment with DT for 3 days indeed resulted in gradual somatotrope ablation with a final loss of 80-90% 1 week later.
Results: The stem/progenitor cell-clustering side population expanded after injury, concordant with an increase in Sox2 + stem/progenitor cells. In addition, folliculo-stellate cells, previously designated as pituitary stem/progenitor cells and significantly overlapping with Sox2 + cells, also increased in number. Remarkable was the appearance of Sox2 + cells that contained GH. GH + cells considerably regenerated during the months after the toxin. Double Sox2 + /GH + cells are observed throughout the regenerative period, which could support the recovery of somatotropes from stem/progenitor cells, further supported by 5-ethynyl-2’-deoxyuridine (EdU) pulse-chase lineage tracing.
Conclusion: This study shows that the adult pituitary gland is capable of regeneration and suggests that the stem/progenitor cells are involved in the repopulation of the gland.
In various adult organs, stem cells play a significant role in tissue repair after injury. The pituitary gland has enormous plasticity in terms of regulation of hormone-producing cells, for example the rise in lactotrope number during pregnancy and lactation. Whether the pituitary gland is capable of cell regeneration after cell loss at adult age is not clear.
Sox2 + cells localized in the marginal zone around the pituitary cleft, which is a remnant of Rathke's pouch, are believed to be pituitary progenitors. They do not normally express genes encoding pituitary hormones. The authors used a neat system where diphtheria toxin (DT) receptor is expressed in somatotropes only, so that DT treatment will ablate somatotropes only. Three days after DT-induced somatotrope ablation, the ‘stem cell – side population’, artificially defined as ‘non-Sca-1-immunoreactive cells of the Hoechst dye effluxing side population’, expanded and the number of Sox2 + cells increased. In contrast to the situation before ablation, these Sox2 + cells were proliferating. In addition, folliculo-stellate cells were proliferating and SOX2 + /GH + cells started to appear. The authors suggest therefore that GH-positive cells arise from the increased number of stem/progenitor cells in the pituitary after injury.
There are some pitfalls, however. Variation of Cre expression commonly exists and will result in variable DT-receptor expression and variable ablation of somatotropes, and also somatolactotropes. Indeed, only 80% of somatotropes and 50% of lactotropes were ablated. In this context, we do not know whether the SOX2 + /GH + cells reflect trans-differentiated hormone-producing cells that started to express SOX2 or really reflect newborn GH-expressing cells developing from SOX2 + /GH - progenitors. The authors conclude that the evidence supports a role for stem/progenitor cells in the regeneration process of somatotropes at adult age. Indeed, it is not proven but only provides some evidence towards this conclusion. However, it is a good start. Whether we will ever use pituitary stem cells for treatment, for example in POU1F1 deficiency, remains to be seen. Most forms of multiple pituitary hormone deficiency would require allogenic donor cells and normal hypothalamic function for such treatments to work.

MEN1 gene replacement therapy reduces proliferation rates in a mouse model of pituitary adenomas
Walls GV, Lemos MC, Javid M, Bazan-Peregrino M, Jeyabalan J, Reed AA, Harding B, Tyler DJ, Stuckey DJ, Piret S, Christie PT, Ansorge O, Clarke K, Seymour L, Thakker RV
Academic Endocrine Unit, Nuffield Department of Clinical Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Headington, Oxford, UK
Cancer Res 2012;72:5060-5068
Background: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by multiple endocrine tumors, such as adenomas of the parathyroids, anterior pituitary and adrenals, and neuroendocrine tumors of the pancreas, duodenum, stomach and sometimes lungs or thymus, and is caused by haploinsufficiency of the tumor suppressor gene MEN1 , encoding the menin protein. Treatment of these neuroendocrine tumors (NET) is more difficult than of sporadic NETs since tumors of MEN1 patients tend to metastasize more frequently and have a more invasive behavior. Therefore, there is clinical need for better treatment. In vitro studies have shown that expression of Men1 in tumor cell lines partially re-establishes the wild-type phenotype by reducing proliferation and increasing apoptosis. This is the first study to use Men1 gene replacement in mice, and this resulted in a reduction of proliferation and tumor growth.
Methods: Replication-deficient adenovirus serotype 5 was used to generate the construct expressing the murine Men1 cDNA. Men1 heterozygous mice that develop NET were used. Delivery of adenoviral vectors was achieved by transauricular intratumoral injection of Men1. rAd5 and GFP. rAd5 (5 × 10 10 viral particles of both). Efficiency of transauricular intratumoral injection was assessed by MRI scanning and visualization of the GFP.
Results: Men1 cDNA was introduced in the replication-deficient adenovirus serotype 5 (rAd5) to create Men1 .rAd5. In vitro studies showed that the menin protein was produced in Men1 .rAd5 infected Men1 -/- murine embryonic fibroblasts (MEFs) and that pituitary tumor cell lines were infected with increasing concentration of Men1. rAd5 viral particles. Tumors of animals treated with Men1. rAd5 showed decreased proliferation without changes in apoptosis. However, tumor size did not change.
Conclusions: This study establishes the proof of concept that Men1 gene replacement in vivo reduces cell proliferation of anterior pituitary adenomas that develop in Men1 +/- mice.
This elegant paper by Walls et al. is an important advancement for the development of MEN1 gene therapy in pituitary adenomas. This work may open the way for treatment by adenovirus genemediated delivery of other MEN1-related tumors, for example the pancreas or other pituitary tumors.
New knowledge – SOX2

SOX2 regulates the hypothalamic-pituitary axis at multiple levels
Jayakody SA, Andoniadou CL, Gaston-Massuet C, Signore M, Cariboni A, Bouloux PM, Le Tissier P, Pevny LH, Dattani MT, Martinez-Barbera JP
Neural Development Unit, Institute of Child Health, and Department of Cell and Developmental Biology, University College London, London, UK
J Clin Invest 2012;122:3635-3646
Background: Sex-determining region Y (SRY) box 2 (SOX2) haploinsufficiency causes hypogonadotrophic hypogonadism. Associated features include developmental abnormalities of the eye such as anophthalmia, brain abnormalities such as hypoplasia of the corpus callosum and hypothalamic hamartoma, sensorineural hearing loss and esophageal atresia. Rarely, GH deficiency may be a feature. To date, the presence of gonadotrophin deficiency in the face of the normal secretion of other pituitary hormones remains unexplained. Sox2 is expressed in both the hypothalamus as well as Rathke's pouch during development, but not in the infundibulum or posterior lobe. The authors aimed to investigate the pathogenesis of hypogonadotrophic hypogonadism in SOX2-haploinsufficient patients.
Methods: Given that Sox2 ablation leads to early embryonic lethality soon after implantation, preventing the study of its function during organogenesis, the authors conditionally deleted Sox2 in mice, using a strategy whereby the Hesx1 Cre/+ mouse line was crossed with Sox2 fl/fl animals; this strategy led to ablation of Sox2 within the pituitary.
Results: First, absence of SOX2 in the developing Rathke's pouch of conditional embryos led to severe anterior lobe hypoplasia with drastically reduced expression of the pituitary-specific transcription factor POU class 1 homeobox 1 (POU1F1) as well as severe disruption of somatotrope and thyrotrope differentiation. In contrast, corticotropes, rostral-tip POU1F1 -independent thyrotropes, and, interestingly, lactotropes and gonadotropes were less affected. Although there was no statistically significant variation from the expected mendelian ratios during embryogenesis (9.5-18.5 dpc), genotyping of postnatal mice from birth to 3 weeks failed to identify any viable Hesx1 Cre/+ ;Sox2 fl/fl mice, showing that deletion of Sox2 using the Hesx1-Cre line leads to neonatal/perinatal death. Second, the authors identified a requirement for Sox2 in normal proliferation of periluminal progenitors; in its absence, insufficient precursors were available to produce all cell lineages of the anterior pituitary. Differentiated cells derived from precursors exiting cell cycle at early stages, including corticotropes, rostral-tip thyrotropes, and gonadotropes, were generated, while hormone-producing cells originating from late-born precursors, such as somatotropes and POU1F1 -dependent thyrotropes, were severely reduced. Finally, the authors reported a lack of GnRH immunoreactivity in the median eminence of Hesx1 Cre/+ ;Sox2 fl/fl mice as compared with controls at 18.5 dpc, whereas the expression of Ghrh, Nr5a1, Avp and Oxt was similar to controls. Additionally, there was a significant reduction of GnRH neurons in the Hesx1 Cre/+ ;Sox2 fl/fl mice at all stages of development (12.5-18.5 dpc), as GnRH neurons migrate from the olfactory epithelium to the brain, suggesting a possible reduction in neurogenesis of GnRH neurons. In keeping with the murine data, 2 previously characterized patients with SOX2 haploinsufficiency and associated hypogonadotrophic hypogonadism had a measurable response to gonadotropin-releasing hormone (GnRH) stimulation, suggesting that it is not the absence of gonadotrope differentiation, but rather the deficient hypothalamic stimulation of gonadotropes, that underlies typical hypogonadotrophic hypogonadism.
Conclusion: Sox2 is required for proliferation of Rathke's pouch progenitors and expansion of the anterior pituitary during embryogenesis. Additionally, whilst Sox2 ablation does not affect the development of the neuroendocrine hypothalamus, it appears to be essential for the generation of GnRH neurons, accounting for the rather specific hypogonadotrophic hypogonadism observed in individuals with haploinsufficiency of SOX2.
Previous data using hypomorphic and Sox2 conditional alleles had revealed that severe reduction or complete removal of Sox2 within the developing neural tube does not cause gross morphological defects in the brain or spinal cord, possibly due to redundancy with Sox1 and Sox3. These genetic approaches have, however, revealed an essential role for SOX2 in the maintenance of neurons in specific brain regions, proliferation and/or maintenance of neural stem cells, and neurogenesis.
In this paper, Jayakody et al. used a conditional deletion of Sox2 in the hypothalmo-pituitary region to show that, although at 8.5-10.5 dpc there were no gross morphological defects in the Rathke's pouch (RP) of the mutants, by 18.5 dpc there was a fully penetrant phenotype of severe anterior pituitary hypoplasia in all embryos analyzed, often with ectopically located anterior pituitary tissue within the oropharyngeal ectoderm. These data suggest that in the absence of Sox2 , normal early induction of RP occurs, but there is failure of subsequent AP development and expansion, leading to a very small and partially ectopic anterior lobe. Additionally, while terminal differentiation of somatotropes and mature thyrotropes was severely disrupted in the mutants, corticotropes/melanotropes, thyrotropes/gonadotropes and lactotropes were less affected. In keeping with these data, there was a dramatic reduction in Pou1f1 + cells from 12.5-18.5 dpc in the Hesx1 Cre/+ ;Sox2 fl/fl mutant animals. Prop1 expression was reduced in the mutant pituitaries at 12.5 dpc. Rostral-tip Pou1f1-independent thyrotropes were normal, as were corticotropes. These are the first two differentiated cell types to be specified in the early developing anterior lobe. The relative sparing of lactotropes in the mutant animals remains puzzling, given that lactotrope specification is dependent on Pou1f1. The data also suggest that the mutant phenotype is related to the timing of cell-cycle exit; rostraltip thyrotropes, corticotropes and gonadotropes, whose precursors become post-mitotic from 11.5 dpc, are least affected, whereas the later-differentiating thyrotropes and somatotropes are almost absent.
The surprising finding of this study is that whilst the hypoplastic pituitary is a consequence of loss of Sox2 within the developing RP, pituitary stimulation with high-dose GnRH resulted in LH and FSH production, suggesting that the hypogonadotrophic hypogonadism observed in human patients is due to hypothalamic GnRH deficiency. This study underlines the importance of murine transgenesis in understanding human phenotypes, and also demonstrates the complexity of hypothalamo-pituitary development, whereby the same gene can act at multiple levels within the HP axis during different stages of development. However, the study also emphasizes the differences that are apparent between the murine and human phenotypes, for example, thyrotropes are severely reduced in the mutant mice whereas hypothyroidism has not been documented to date in humans. Additionally, humans with SOX2 haploinsufficiency have slow-growing pituitary tumors, whereas this has not been observed in the murine model [ 7 ]. Rather, the next paper shows that Sox2 haploinsufficiency may protect mice from developing pituitary tumors.

p27 Kip1 directly represses Sox2 during embryonic stem cell differentiation
Li H, Collado M, Villasante A, Matheu A, Lynch CJ, Canamero M, Rizzoti K, Carneiro C, Martinez G, Vidal A, Lovell-Badge R, Serrano M
Tumor Suppression Group, Spanish National Cancer Research Centre, Madrid, Spain
Cell Stem Cell 2012;11:845-852
Background: Three main factors, called the Yamanaka factors, Oct4, Klf4 and Sox2, are required for reprogramming of cells to induce pluripotent stem cells. Interestingly, mouse embryonic fibroblast (MEFs) that lack the tumor suppressor p27 kip1 do not require Sox2 to be reprogrammed into pluripotent stem cells. p27 kip1 is a tumor suppressor gene that when mutated leads to a multiple endocrine neoplasia (MEN) syndrome, characterized by pituitary tumors. The observation that absence of p27 kip1 leads to reprogramming without the key stem cell reprogramming factor Sox2, made the authors investigate the relationship between p27 Kip1 and Sox2. They found that p27 Kip1 negatively regulates expression of Sox2, and so may induce proliferation and promote tumorigenesis.
Methods: The authors performed cell reprogramming experiments in primary p27-null mouse embryo fibroblasts. ChiP, RNA quantification and protein assays were used to identify the binding of p27 Kip1 regulatory regions in the Sox2 enhancer and to assess transcriptional regulation of Sox2. p27 Kip1 null mice were crossed with Sox2 +/- mice to assess rescue of the phenotype of the p27 Kip1 null mice.
Results: Cells obtained from p27 kip1 null mice could be converted into induced pluripotent stem cells (iPSC) without the reprogramming factor Sox2. Sox2 protein was increased in cells lacking p27 kip1 , suggesting that p27 kip1 negatively regulates Sox2 expression. The authors demonstrated by ChiP that p27 kip1 was able to bind to a 4Kb Sox2-distal enhancer, together with a repressive complex p130-E2F4-SIN3A. Moreover, overexpression of p27 Kip1 resulted in a reduction of Sox2 expression. Importantly, in vivo, haploinsufficiency of Sox2 in the p27 Kip1 null mice resulted in normalization of some of the features, notably the gigantism, pituitary hyperplasia, pituitary tumor formation and retinal dysplasia.
Conclusions: These experiments provide genetic and biochemical support for a direct regulation of the developmental transcription factor Sox2 by p27 Kip1 .
In this elegant piece of work, Han Li and colleagues identify regulation of the progenitor/stem cell factor Sox2 by the tumor suppressor protein p27 Kip1 . Germline mutations of the p27 Kip1 gene, also known as CDNK1B , are responsible for a subset of MEN syndromes, notably characterized by pituitary tumors [ 8 ]. p27 Kip1 null mice have increased body size, organ hyperplasia, pituitary tumors and retinal dysplasia. This study establishes a previously unknown relationship between p27 kip1 and Sox2 and shows that absence of p27 Kip1 leads to de-repression of Sox2. The authors show that haploinsufficiency of Sox2 is able to rescue the MEN phenotype of p27 kip1 null mice. Sox2 is a crucial gene in the development of multiple tissues, and is also required for maintaining the pituitary progenitor/stem cell population and normal hypothalamic-pituitary development, as outlined in some of the other papers in this and the previous year's chapters. Mutations in SOX2 cause a spectrum of hypothalamic-pituitary abnormalities ranging from hypogonadotrophic hypogonadism to septo-optic dysplasia. This paper presents the first genetic and molecular evidence for a relationship between Sox2 and p27 kip1 . There may be a ‘ying-yang’ balance of effects, where too little SOX2 results in pituitary hormone deficiency and too much SOX2 results in pituitary tumors.
Important for clinical care – outcome of pituitary disease

Outcome of surgical treatment of 200 children with Cushing's disease
Lonser RR, Wind JJ, Nieman LK, Weil RJ, DeVroom HL, Oldfield EH
Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA
J Clin Endocrinol Metab 2013;98:892-901
Background: Factors influencing the outcome of surgical treatment of pediatric Cushing's disease (CD) have not been fully established.
Methods: The authors assessed the outcome of surgery for pediatric CD and factors influencing the outcome in 200 consecutive patients with CD treated at a single tertiary center at the National Institutes of Health (NIH) from 1982 through 2010.
Results: Mean age at symptom development was 10.6 ± 3.6 years (range 4.0-19.0) and at surgery was 13.7 ± 3.7 years. 27 patients (13%) had prior surgery at another institution. MRI identified adenomas in 97 patients (50%) and accurately defined localization (in 99%), which was more accurate than inferior petrosal sinus sampling (accurate in 72%). 195/200 patients (98%) achieved remission after surgery (189 (97%) were hypocortisolemic; 6 (3%) were eucortisolemic postoperatively). Identification of an adenoma at surgery, immunohistochemical ACTH-producing adenoma, and noninvasive ACTH adenoma were associated with initial remission (p < 0.05). Younger age, smaller adenoma, and absence of cavernous sinus wall or other dural invasion were associated with long-term remission (p < 0.05). A 9 a.m. serum cortisol <1 μg/dl after surgery had a 96% positive predictive value for lasting remission.
Conclusion: Pituitary surgery for CD in children can be safe, effective, and durable. Undetectable cortisol concentration postsurgery predicts lasting remission. Lasting remission is associated with younger age at surgery, smaller adenomas, and lack of dural invasion.
This is a beautiful report of prospective data collection and evaluation of pre- and postoperative management of pediatric Cushing's disease in a single center. 200 patients seen between 1982 and 2010 were included. In line with previous reports, there was predominance of males (F:M 0.8:1) in patients <15 years, but more females among older patients (3.8:1) [ 9 ]. An astonishing 98% of patients reached remission immediately after surgery and 90% remained in remission for at least 10 years, the highest success rate reported so far [ 10 , 11 ]. Rapid weight gain and poor growth were the most frequent symptoms, followed by the presence of dorsal cervical or supraclavicular fat pads. MRI with SGPR (standard spin echo sequences and spoiled gradient recalled) acquisition sequences improved the poor detection of adenomas from 50 to 65%. IPPS correctly predicted the site of the adenoma in 70%, similar to reports of other series [ 9 , 12 ].
Adenoma size was on average 7.9 mm and invasion occurred in 20%. In all patients, trans-sphenoidal adenomectomy, or partial hypophysectomy if no adenoma was identified, was performed. In 13%, no adenoma was identified and an ACTH-producing tumor was only found on histology in 30% of these cases. Not surprisingly, these children had the worst outcome. As reported before, a postoperative morning cortisol concentration <1 μg/dl predicted lasting remission with an accuracy of 96%. Interestingly, only 5% developed DI and only 3% symptomatic hyponatremia. Unfortunately, the data did not include postoperative assessment of pituitary function.
This paper shows that outcome is better for younger children and for non-invading tumors, so our next task is to reduce the time between the first symptoms and the diagnosis. This study also highlights the value of prospective databases to audit the management and outcome of patients with rare diseases, both at institutional and national levels. Other examples are the UK National Diabetes Audit and the European DSD database.

Recovery of the hypothalamic-pituitary-adrenal axis in children and adolescents after surgical cure of Cushing's disease
Lodish M, Dunn SV, Sinaii N, Keil MF, Stratakis CA
Section on Endocrinology and Genetics Program on Developmental Endocrinology and Genetics, National Institutes of Health, Bethesda, MD, USA
J Clin Endocrinol Metab 2012;97:1483-1491
Background: The hypothalamic-pituitary-adrenal axis (HPAA) recovers slowly after trans-sphenoidal surgery (TSS) for Cushing's disease (CD), but data on recovery times are not available.
Methods: The authors aimed to assess time to recovery of the HPAA after TSS in children with CD. 57 patients with CD (6-18 years, mean 13.0 ± 3.1 years) were given a standard regimen of glucocorticoid tapering after TSS. Synacthen stimulation tests were performed 6-monthly for up to 36 months. Age, sex, pubertal status, body mass index, length of disease, midnight cortisol, and urinary free cortisol at diagnosis were analyzed for effects on recovery. Primary outcome was complete recovery of the HPAA, defined by a cortisol concentration >18 μg/dl in response to 250 μg ACTH.
Results: HPAA recovered in 75% of patients, with 67 and 95% recovering by 12 and 18 months, respectively. The mean time to recovery was 12.6 ± 3.3 months. Survival analyses estimated a 75% chance of recovering within 14 months. A cut-off of at least 10-11 μg/dl of cortisol as the peak of ACTH stimulation testing at 6 months after TSS yielded the highest sensitivity (70-80%) and specificity (64-73%) to predict full recovery of the HPAA at 12 months. Importantly, 2 of the 4 patients who recovered fully within 6 months had recurrent CD.
Conclusions: The standardized tapering regimen used for glucocorticoid replacement after TSS led to recovery of the HPAA in most patients within the first postoperative year. Multiple factors may affect this process, but an early recovery may indicate disease recurrence.
Cortisol overproduction in Cushing's disease results in negative feedback and suppression of hypothalamic CRH production, resulting in adrenal insufficiency after removal of the ACTH-producing tumor. Adrenal function needs to be followed up not only to assess the appropriate time to discontinue HC treatment, but also to detect patients with recurrent disease. Although this study was not prospective, has missing data, and children with early recurrence were excluded, it still gives valuable data on the postsurgical HPAA in a large number of patients with Cushing's disease. Good HPA function was defined as a cortisol response of 18 µg/dl (500 nmol/l) after the non-physiological high-dose (250 µg ACTH) Synacthen test. 10% recovered within 6 months, and 60% recovered within a year. A peak cortisol cut-off of >10-11 µg/ml predicted for a normal response 6 months later with a reasonable specificity and sensitivity. This time scale is similar to reports of recovery of pituitary GH and TSH axes. An important message of this paper is that HPA recovery within 6 months may predict relapse of Cushing's disease.
Interestingly, hydrocortisone replacement schedule consisted of only 2 doses per day, starting at a dose of 8-12 mg/m 2 /day, followed by a rapid reduction after 4 months of 2.5 mg/day every 4-6 weeks and omission of the afternoon dose as soon as possible. After passing the ACTH test, emergency hydrocortisone treatment was advised for a further 6 months. This tapering regimen required adjustment of dose in only 6% of cases (patients were warned to expect symptoms of adrenal insufficiency during the first 3-4 months after surgery) and no hospitalization for adrenal insufficiency was required in any patient, suggesting that this tapering regimen is safe. Many centers use three times daily dosing and stop further dose reduction when a physiological dose is reached. As the authors point out, it is not clear how their empirical tapering regimen affected adrenal recovery.

Xanthogranuloma, Rathke's cyst, and childhood craniopharyngioma: results of prospective multinational studies of children and adolescents with rare sellar malformations
Muller HL, Gebhardt U, Faldum A, Warmuth-Metz M, Pietsch T, Pohl F, Calaminus G, Sorensen N
Department of Pediatrics, Klinikum Oldenburg, Oldenburg, Germany
J Clin Endocrinol Metab 2012;97:3935-3943
Background: No consensus guidelines exist for the management of craniopharyngioma (CP), Rathke's cyst (RC), and xanthogranuloma (XG) which are closely related, rare, sellar masses. This study assessed clinical manifestations, treatment and outcome of patients with such masses.
Methods: The authors performed a multicenter surveillance trial of patients <18 years with a histological diagnosis of CP, XG, or RC. 117 patients with CP, 14 with XG, and 14 with RC were included.
Results: The 5-year OS (overall survival) rates were 1.00 ± 0.00 in RC and XG and 0.97 ± 0.02 in CP. The 5-year EFS (event-free survival) rates were 0.85 ± 0.10 in RC, 1.00 ± 0.00 in XG, and 0.50 ± 0.05 in CP. Surgical resection of XG resulted in complete remission without recurrence. Recurrences occurred in RC (14%) and CP (59%). CP leads most often to severe hypothalamic sequelae such as obesity and others affecting QoL, mostly due to presurgical involvement (59%) and postsurgical lesions (44%) of posterior hypothalamic structures. Centers with lower neurosurgery patient loads used more radical surgical approaches to treat CP, resulting in higher rates of obesity and reduced QoL. Despite 46% anterior hypothalamic involvement, severe obesity is not encountered in XG.
Conclusion: The treatment of choice in XG and RC is radical surgery. In CP, less radical surgical approaches that preserve hypothalamic structures are recommended. Regular imaging during follow-up is recommended for CP and RC because of frequent relapses. Treatment of patients with sellar masses should be confined to experienced multidisciplinary teams.
Craniopharyngioma (CP), Rathke's cyst (RC), and sellar xanthogramuloma (XP) are closely related lesions. CPs arise from neoplastic transformation of ectodermal cell remnants of Rathke's pouch and the craniopharyngeal duct. RCs are also Rathke's pouch-derived epithelial lesions. XG of the sellar region are composed of cholesterol clefts, macrophages, chronic inflammatory cellular reaction, and hemosiderin deposits. Outcomes of various treatment modalities for CP are emerging, allowing for development of clinical guidelines, but very little data of long-term follow-up exist for RC and XP, hence the importance of this prospective study. CPs and RCs are frequently seen in clinical practice, but XG is rare.
Unexpected findings were that hydrocephalus was seen in 15% of patients with RC, that duration of symptoms was longest in XG, and that XG commonly invaded the hypothalamus (in 48% of cases) although not as deeply as CP. Surgical outcomes were excellent for RC and XG but, as expected, were less favorable for CP. Despite frequent hypothalamic invasion, obesity was not a common complication of XG, even after surgical removal, in contrast to CP.
In this study, only patients with a histological diagnosis and surgical removal of RC, CP or XG were included. RC are commonly found as incidentalomas and small RC are believed not to require treatment [ 13 , 14 ]. In the present study, the causal relationship between symptoms and the sellar masses could not be assessed, nor the outcome of conservative treatment and the question whether RC requires treatment, and if so, the optimal timing of this. Further prospective studies of the management of patients with sellar masses are needed to address these questions and to enable the development of clinical guidelines for the management of XG and RC.
New knowledge of pituitary tumors

A novel mutation in the upstream open reading frame of the CDKN1B gene causes a MEN4 phenotype
Occhi G, Regazzo D, Trivellin G, Boaretto F, Ciato D, Bobisse S, Ferasin S, Cetani F, Pardi E, Korbonits M, Pellegata NS, Sidarovich V, Quattrone A, Opocher G, Mantero F, Scaroni C
Department of Medicine, Endocrinology Unit, University of Padova, Padova, Italy
PLoS Genet 2013;9:e1003350
Background: Multiple endocrine neoplasia syndrome type 4 is an autosomal dominant syndrome characterized by a varying combination of tumors affecting at least two endocrine organs. It resembles MEN1, but patients have no mutation in MEN1. Recently p27 Kip1 , a cycling-dependent kinase 1, has been shown to have tumor suppressor activity because it inhibits multiple cyclin-dependent kinases, stopping cells progressing from G1 to S phase. Moreover, germline mutations in the gene CDKN1B that encodes p27 kip1 are responsible for multiple endocrine neoplasia (MEN) type 4 syndromes. This study identifies for the first time a 4 base-pair deletion in the upstream open reading frame sequence (uORF) of the CDKN1B gene that affects transcriptional levels of p27 kip1 in a patient with pituitary and adrenal neoplasia.
Methods: 25 patients with MEN1-related symptoms were screened for mutations in CDKN1B. Sequencing of the open reading frame, exon-intron boundaries and upstream sequences identified no mutations, apart from 1 patient with a 4 base-pair deletion in the uORF. Functional studies (dual luciferase assay, site-directed mutagenesis, and polysome profiling) showed an effect of the mutation on transcriptional levels of p27 kip1 .
Results: Transcription and translation of some genes can be influenced by sequences termed upstream open reading frame, uORFs. These elements can affect the translational machinery. A small 4 base-pair deletion in the p27 Kip1 uORF was identified by sequencing, so the question arose whether this deletion can affect translation of the protein. Initial studies demonstrated that the level of mRNA did not change, indicating that transcription occurs normally. However, luciferase assays showed lower luciferase levels for the construct compared to wild type. Moreover, in vitro expression of the mutant uORF driving p27 kip1 in HEK 293 cells led to a smaller amount of generated protein. This suggests that the 4 base-pair deletion is sufficient to affect translation of the protein.
Conclusions: The deletion identified in this study in the p27 Kip1 uORF is a novel example of a uORF-affecting mutation that can lead to endocrine neoplasia. The data clearly show a negative effect of the 4 base-pair deletion on translation of the tumor suppressor p27 kip1 .
MEN4 has been described as a new type of multiple endocrine neoplasia [ 15 ]. The phenotype resembles MEN1, and the most common phenotypic features are parathyroid and pituitary adenomas. So far, heterozygous mutations at various residues of the tumor suppressor gene p27 Kip1 have been found in patients with MEN4. As discussed earlier in this chapter, p27 Kip1 negatively regulates the stem cell factor SOX2 [ 16 ]. This paper shows for the first time how mutations in upstream open reading frame regulatory regions of p27 Kip1 affect translation of the protein, and are likely the cause of MEN4 in these patients. This paper provides novel insights into the role of uORFs in protein translation in general and in the pathogenesis of MEN4 specifically. It sets the road ahead for the investigation of mutations in uORFs of other genes involved in pituitary disease as it is foreseen that mutations in regulatory regions will affect protein production and so influence pituitary development and homeostasis.
New model

Developmental analysis and influence of genetic background on the Lhx3 W227ter mouse model of combined pituitary hormone deficiency disease
Prince KL, Colvin SC, Park S, Lai X, Witzmann FA, Rhodes SJ
Departments of Cellular and Integrative Physiology, Indiana University-Purdue University, Indianapolis, IN, USA
Endocrinology 2013;154:738-748
Background: Although a number of genes have been implicated in the etiology of congenital hypopituitarism, the majority of these disorders remain unexplained. Little is known about their etiology, especially the developmental profiles and the influence of genetic background on disease progression. Animal models for Combined Pituitary Hormone Deficiency (CPHD) provide valuable tools to investigate disease mechanisms and inform diagnostic and treatment protocols. Lhx3/LHX3 is a transcription factor that is expressed during the early stages of pituitary and CNS development and plays essential roles in cell differentiation within the anterior pituitary, and for the differentiation of neurons within the brain and spinal cord. Mutations in LHX3 are associated with severe CPHD in addition to sensorineural hearing loss and a rigid cervical spine resulting in limited head rotation.
Methods: The authors aimed to investigate hormone production during pituitary development and the influence of genetic background on phenotypic severity in the mouse model of an Lhx3 mutation (Lhx3 W227ter/W227ter ). In humans, this mutation is associated with CPHD without cervical spine rigidity. In previous studies, knock-in of the W227ter mutation in a murine model of mixed background (129/ Sv + C57BL/6) led to the generation of a mouse model of CPHD without any CNS defects. Through breeding of Lhx3 W227ter/+ mice of mixed background (purity 98.4%); the Lhx3 W227ter/W227ter genotype was placed onto the 129/Sv and C57BL/6 backgrounds. The mice were then analyzed using immunohis-tochemistry at different stages. Proteomic analysis of wild-type and mutant pituitaries was performed.
Results: Lhx3 W227ter/W227ter embryos had deficiencies of ACTH, α-glycoprotein subunit, GH, PRL, TSHβ, and LHβ during prenatal development. Furthermore, mutant mice had a reduction in the critical pituitary transcriptional activator-1 (PIT1/POU1F1). Intriguingly, the genetic background affected viability: whereas Lhx3 W227ter/W227ter animals were found in the expected frequencies in the C57BL/6 background, homozygous animals were not viable in the 129/Sv genetic background. The hormone marker and PIT1 reductions observed in Lhx3 W227ter/W227ter mice on a mixed background were also seen in the separate strains but in some cases were more severe in 129/Sv. Proteomic analysis revealed lower levels of PRL, pro-opiomelanocortin (ACTH), and α-glycoprotein subunit proteins in Lhx3 W227ter/W227ter mice. A number of protein networks were affected in the mutated animals and included cell cycle, cellular maintenance and protein translation networks.
Conclusion: These data show that hormone deficiencies are manifest in early prenatal stages in this CPHD model system. Furthermore, as is noted in human disease, genetic background impacts the phenotypic outcome of these monogenic endocrine diseases.
This paper reports functional studies of a mutation in LHX3 that is associated with a pituitary-specific phenotype, with no abnormality of neck rotation, unlike other described LHX3 mutations. The W227ter mutation is the most carboxy-terminal mutation in LHX3 [ 17 ], and the pituitary-specific phenotype underlines the importance of the carboxy-terminus in pituitary development. Previous studies showed that knock-in of this mutation in a mixed murine background led to a pituitary-specific phenotype [ 18 ]. In this study, the authors back-crossed the Lhx3 W227ter/+ heterozygous mice on a mixed background to generate mice with two pure backgrounds (C57BL/6 and 129/Sv). Intriguingly, the phenotypes were different, being more severe in the 129/Sv mice. The latter were not viable, unlike those in the C57BL/6 background, which were born in the expected ratios. This is consistent with data in humans, where phenotypes can vary markedly, even with the same genetic mutation. Importantly, this paper also shows that the mutant animals manifested both structural and hormonal pituitary abnormalities prenatally, with reduced Pit1/Pou1f1 expression.
Proteomic analysis revealed not only changes in hormonal expression, but also in genes implicated in cell cycle progression, cellular maintenance, and protein translation networks. Surprisingly, in view of increased apoptosis and pituitary hypoplasia in Lhx3 -/- and Lhx3 W227ter/W227ter mutants, 17 proteins associated with apoptosis were downregulated. It is important to note that proteomic analysis was performed in adult animals, and the results therefore need to be interpreted cautiously.
Review of the year

Cushing syndrome in pediatrics
Stratakis CA
Section on Endocrinology and Genetics, Program in Developmental Endocrinology and Genetics, Eunice Kennedy
Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
Endocrinol Metab Clin North Am 2012;41:793-803
Cushing syndrome is characterized by truncal obesity, growth deceleration, skin changes, muscle weakness, and hypertension. Cushing syndrome in childhood usually results from the exogenous administration of glucocorticoids. This article presents the causes and discusses the treatment of endogenous Cushing syndrome. It also discusses the clinical and molecular genetics of inherited forms of this syndrome. Cushing syndrome needs to be diagnosed and treated properly when first recognized; improper treatment can turn this otherwise completely curable disorder into a chronic ailment. Barriers to optimal care of the pediatric patient with Cushing syndrome are discussed.
This is yet another excellent review by Stratakis that discusses in detail symptomatology, underlying genetic causes, diagnostic tests and surgical and medical treatment of Cushing syndrome. Diagnostic testing including specificity and sensitivity of tests, the importance of the correct sequence of tests and the need to avoid imaging before a biochemical diagnosis has been reached, are discussed. This review not only serves as support for the clinician investigating or excluding Cushing syndrome, but can also be used as the basis for teaching.
Food for thought

Kallmann syndrome
Smith N, Quinton R
Royal Victoria Infirmary, Newcastle upon Tyne, UK
In the era of PREMS and PROMS (‘patient reported experience measures’ and ‘patient reported outcome measures’, respectively), this account of an adult who had a delayed diagnosis of Kallmann syndrome at the age of 22 years is well worth reading. It describes the long road before the correct diagnosis was made and the psychological consequences of the delay. Hopefully, increased awareness of Kallmann syndrome among our non-endocrine colleagues has already increased the number of referrals made at the correct age and to the correct specialist, with hopefully improved long-term outcomes.
1. Gutnick A, Blechman J, Kaslin J, Herwig L, Belting HG, Affolter M, et al: The hypothalamic neuropeptide oxytocin is required for formation of the neurovascular interface of the pituitary. Dev Cell 2011;21:642-654.
2. Dores RM, Baron AJ: Evolution of POMC: origin, phylogeny, posttranslational processing, and the melanocortins. Ann NY Acad Sci 2011;1220:34-48.
3. McCabe MJ, Gaston-Massuet C, Tziaferi V, Gregory LC, Alatzoglou KS, Signore M, et al: Novel FGF8 mutations associated with recessive holoprosencephaly, craniofacial defects, and hypothalamo-pituitary dysfunction. J Clin Endocrinol Metab 2011;96:E1709-1718.
4. Raivio T, Avbelj M, McCabe MJ, Romero CJ, Dwyer AA, Tommiska J, et al: Genetic overlap in Kallmann syndrome, combined pituitary hormone deficiency, and septo-optic dysplasia. J Clin Endocrinol Metab 2012;97:E694-699.
5. Avbelj Stefanija M, Jeanpierre M, Sykiotis GP, Young J, Quinton R, Abreu AP, et al: An ancient founder mutation in PROKR2 impairs human reproduction. Hum Mol Genet 2012;21:4314-4324.
6. Matsumoto S, Yamazaki C, Masumoto KH, Nagano M, Naito M, Soga T, et al: Abnormal development of the olfactory bulb and reproductive system in mice lacking prokineticin receptor PKR2. Proc Natl Acad Sci USA 2006;103:4140-4145.
7. Alatzoglou KS, Andoniadou CL, Kelberman D, Buchanan CR, Crolla J, Arriazu MC, et al: SOX2 haploinsufficiency is associated with slow progressing hypothalamo-pituitary tumours. Hum Mutat 2011;32:1376-1380.
8. Marinoni I, Pellegata NS: p27kip1: a new multiple endocrine neoplasia gene? Neuroendocrinology 2011;93:19-28.
9. Savage MO, Chan LF, Grossman AB, Storr HL: Work-up and management of paediatric Cushing's syndrome. Curr Opin Endocrinol Diabetes Obes 2008;15:346-351.
10. Storr HL, Chan LF, Grossman AB, Savage MO. Paediatric Cushing's syndrome: epidemiology, investigation and therapeutic advances. Trends Endocrinol Metab 2007;18:167-174.
11. Magiakou MA, Mastorakos G, Oldfield EH, Gomez MT, Doppman JL, Cutler GB Jr, et al: Cushing's syndrome in children and adolescents. Presentation, diagnosis, and therapy. N Engl J Med 1994;331:629-636.
12. Storr HL, Alexandraki KI, Martin L, Isidori AM, Kaltsas GA, Monson JP, et al: Comparisons in the epidemiology, diagnostic features and cure rate by transsphenoidal surgery between paediatric and adult-onset Cushing's disease. Eur J Endocrinol 2011;164:667-674.
13. Famini P, Maya MM, Melmed S: Pituitary magnetic resonance imaging for sellar and parasellar masses: ten-year experience in 2,598 patients. J Clin Endocrinol Metab 2011;96:1633-1641.
14. Trifanescu R, Ansorge O, Wass JA, Grossman AB, Karavitaki N: Rathke's cleft cysts. Clin Endocrinol (Oxf) 2012;76:151-160.
15. Lee M, Pellegata NS: Multiple endocrine neoplasia type 4. Front Horm Res 2013;41:63-78.
16. Li H, Collado M, Villasante A, Matheu A, Lynch CJ, Canamero M, et al: p27 Kip1 directly represses Sox2 during embryonic stem cell differentiation. Cell Stem Cell 2012;11:845-852.
17. Mullen RD, Colvin SC, Hunter CS, Savage JJ, Walvoord EC, Bhangoo AP, et al: Roles of the LHX3 and LHX4 LIM-homeodomain factors in pituitary development. Mol Cell Endocrinol 2007;265-266:190-195.
18. Colvin SC, Malik RE, Showalter AD, Sloop KW, Rhodes SJ: Model of pediatric pituitary hormone deficiency separates the endocrine and neural functions of the LHX3 transcription factor in vivo. Proc Natl Acad Sci USA 2011;108:173-178.
Aurore Carré a , Gabor Szinnai b , Dulanjalee Kariyawasam a , Mireille Castanet c and Michel Polak a
a Paediatric Endocrinology, Gynecology and Diabetology, Hôpital Necker Enfants Malades, AP-HP, INSERM U845, IMAGINE Affiliate, Université Paris Descartes, Centre des Maladies Endocriniennes Rares de la Croissance, Paris, France b Paediatric Endocrinology, University Children's, Hospital Basel, and Department of Biomedicine, University Basel, Basel, Switzerland c Paediatric Endocrinology, Hôpital Charles Nicolle, Rouen, France
Clinical studies for congenital hypothyroidism Generic or not?

Generic levothyroxine compared with synthroid in young children with congenital hypothyroidism
Lomenick JP, Wang L, Ampah SB, Saville BR, Greenwald FI
Department of Pediatrics, Division of Endocrinology, Vanderbilt University School of Medicine, Nashville, TN, USA
J Clin Endocrinol Metab 2013;98:653-658
Background: The authors wished to determine whether a brand-name drug (Synthroid) resulted in better control of congenital hypothyroidism than generic LT 4 .
Methods: This was a 5-year retrospective study conducted at one tertiary care center. Children who were 0-36 months old with congenital hypothyroidism followed up at this center from 2006 to 2011 were treated with either Synthroid exclusively (35 subjects) or generic LT 4 exclusively (27 subjects). The subjects’ TSH and free T 4 measurements and how often their LT 4 dose was adjusted were recorded. The TSH variance between the groups was measured as a primary endpoint. Secondary endpoints were the frequency of LT 4 dose changes and the variance in free T 4 .
Results: There was no difference in TSH SD in the Synthroid group compared with the generic group (median 3.0 vs. 2.2, p = 0.27). Children treated with the generic LT 4 had lower TSH estimated SD (1.35 with 95% confidence interval (CI) (1.194, 1.526)) than the Synthroid group (1.66 with 95% CI (1.536, 1.803)). Similarly, no difference was observed in free T 4 SD between the groups (median 0.29 generic vs. 0.36 Synthroid, p = 0.11), but the generic group had lower free T 4 estimated SD than the Synthroid group (0.216 with 95% CI (0.187, 0.249) vs. 0.298 with 95% CI (0.273, 0.326)). Frequency of LT 4 dosing adjustments was similar between the groups, both in total (median 2.0 for generic vs. 3.0 for Synthroid, p = 0.097) and when adjusted for number of TSH checks (ratio 0.25 generic vs. 0.31 Synthroid, p = 0.45).
Conclusion: In this study of congenital hypothyroidism, generic LT 4 treatment resulted in similar or better control of hypothyroidism compared with Synthroid.

Generic and brand-name L-thyroxine are not bioequivalent for children with severe congenital hypothyroidism
Carswell JM, Gordon JH, Popovsky E, Hale A, Brown RS
Division of Endocrinology, Boston Children's Hospital, Boston, MA, USA
J Clin Endocrinol Metab 2013;98:610-617
Background: In the United States, generic substitution of levothyroxine (LT 4 ) by pharmacists is permitted if the formulations are deemed to be bioequivalent by the Federal Drug Administration. There is however widespread concern beyond the United States that the pharmacokinetic standard used is too insensitive.
Methods: The authors aimed to evaluate the bioequivalence of a brand-name LT 4 (Synthroid) and an AB-rated (AB-rated means that the standard for bioequivalence was met) generic formulation (Sandoz, Princeton, N.J., USA) in children with severe hypothyroidism. This was a prospective randomized crossover study in which patients received 8 weeks of one LT 4 formulation followed by 8 weeks of the other. The setting was an academic medical center. Of 31 children with an initial serum TSH concentration >100 mU/l, 20 had congenital hypothyroidism (CH) (15 of 16 CH patients had severe thyroid dysgenesis or agenesis on imaging) and 11 had autoimmune thyroiditis. The primary endpoint was the serum TSH concentration. Secondary endpoints were the free T 4 and total T 3 concentrations.
Results: The serum TSH concentration was significantly lower after 8 weeks of Synthroid than after generic drug (p = 0.002), but thyroid hormone levels did not differ significantly. The difference in TSH was restricted to patients with CH (p = 0.0005). Patients with CH required a higher LT 4 dose (p < 0.0004) and were younger (p = 0.003) but were not resistant to thyroid hormone. The response to generic vs. brand-name preparation remained significant when adjusted for age.
Conclusion: Synthroid and an AB-rated generic LT 4 are not bioequivalent for patients with severe hypothyroidism due to CH. The authors concluded that it would therefore seem prudent not to substitute LT 4 formulations in patients with severe CH, particularly in those <3 years of age.
At first look, the studies seem to reach opposite conclusions, but they indeed assessed different aspects of the question. Years ago the FDA declared LT 4 preparations to be ‘new’ drugs and required that all existing and future LT 4 products be approved through the new drug application process in order to remain on the US market. However, concerns remained of potentially clinical significance of the accepted differences in a LT 4 concentration of 5% allowed with the most recent pharmacokinetic approach in products designated as bioequivalent by the pharmacokinetic standard used. The authors of the first study concluded that ‘generic LT 4 and Synthroid are at least interchangeable in young children with congenital hypothyroidism’. Such a conclusion is not supported by the data because this retrospective, parallel treatment assessment did not address the interchange of the brand-name product with a rated generics. The only valid conclusion is that contemporary highquality LT 4 products, both generic and brand name, had similar and fairly consistent clinical outcomes from refill to refill when used to treat congenital hypothyroidism.
In the second study, subjects underwent a 16-week, prospective, randomized, open-label, crossover study of treatment with their usual dose of outpatient LT 4 . The study was carefully designed and controlled for many parameters including standardized time of blood sampling, dispensation of medications through a research pharmacy, etc., indeed in a much more robust design than in the first study. At the end of the Synthroid phase of the study, TSH values were significantly lower than after the 8-week generic LT 4 period. This difference in TSH outcomes was most pronounced in the congenital hypothyroidism group. There were no significant differences in FT 4 and TT 3 . Of the 16 patients who were receiving the brand-name product Synthroid at study entry, follow-up TSH values at the end of their Synthroid period were not different from those observed at baseline, indicating consistent TSH outcomes. However the study title and the conclusion on bioequivalence is not completely accurate, as it was not a pharmacokinetic study; so the second study showed us that generic and brand-name L-thyroxine are not clinically interchangeable for congenital hypothyroidism. From a practical point of view we may, as pediatric endocrinologists, alert patients that preparations may be switched at the pharmacy, encourage patients to ask to remain on the same preparation at every pharmacy refill, make sure patients understand the need to have their TSH retested and the potential for dosing readjusted every time their LT 4 preparation is switched.
The battle between generic and brand-name thyroxine is still ongoing! Further research is required to reproduce these results and appropriately assess the ability of pharmacokinetics to predict clinical outcome.
Clinical studies to help understand thyroid development?

A high prevalence of dual thyroid ectopy in congenital hypothyroidism: evidence for insufficient signaling gradients during embryonic thyroid migration or for the polyclonal nature of the thyroid gland?
Wildi-Runge S, Stoppa-Vaucher S, Lambert R, Turpin S, Van Vliet G, Deladoey J
Centre Hospitalier Universitaire Sainte-Justine, Montreal, Que., Canada
J Clin Endocrinol Metab 2012;97:E978-981
Background: Thyroid ectopy results from the failure of the thyroid precursor cells to migrate from the primordial pharynx to the anterior part of the neck. Most ectopic thyroids are revealed by congenital hypothyroidism and present as a single round mass at the base of the tongue, with no other thyroid tissue. The authors aimed to assess the frequency of dual ectopy, with part of the tissue having partially migrated, which they believed occurs more frequently than previously reported.
Methods: To determine the prevalence of dual ectopy, the authors reviewed the pertechnetate scintigraphies of 81 patients with congenital hypothyroidism from thyroid ectopy diagnosed between 2002 and 2011 at their institution.
Results: The authors found 7 cases (9%) of dual ectopy, representing an incidence ranging from 1:50,000 to 1:70,000.
Conclusion: Almost 1 in 10 cases of congenital hypothyroidism due to thyroid ectopy has dual ectopy.
At least two possible explanations may account for dual thyroid ectopy. First, it is conceivable that dual ectopy arises from insufficient signaling gradients in surrounding tissues, which do not allow the primordial thyroid epithelium to develop properly, and that might lead to partial migration of the caudal part of the anlage (primitive thyroid) but not of the apical component, which would result in dual ectopy, even if the thyroid anlage were initially monoclonal. Second, given that the thyroid arises from a small cluster of thyroid follicular cell precursors, which migrates caudally during the first 7 weeks of gestation, dual thyroid ectopy might be a consequence of a migration defect that probably affects two clusters (clones) of primordial thyroid cells to a different degree, leaving none with the potential to reach the normal location and to develop into a bilobed gland. From this, the authors suggested that two populations of cells diverged at an early stage of development, which may arise from insufficient signaling gradients in surrounding tissues during early organogenesis or may indirectly support the polyclonal nature of the thyroid. This interesting article shows us that clinical pediatric endocrinology with well-described patients and developmental biology go together well!

Thyroid Function from Birth to Adolescence in Prader-Willi Syndrome
Sharkia M, Michaud S, Berthier MT, Giguere Y, Stewart L, Deladoey J, Deal C, Van Vliet G, Chanoine JP
Endocrinology and Diabetes Unit, British Columbia's Children's Hospital, University of British Columbia, Vancouver, B.C., Canada
J Pediatr 2013 (E-pub ahead of print)
Background: Prader-Willi syndrome (PWS) is a hypothalamic disorder which raises the question of the possible alterations of the secretion of the pituitary hormones, in addition to the well-known growth hormone deficiency. The authors aimed to describe the response of thyroid-stimulating hormone (TSH) to thyroid-releasing hormone in children and adolescents with PWS, and to compare TSH and total thyroxine (TT 4 ) concentrations measured on neonatal screening for congenital hypothyroidism in children with PWS and controls.
Methods: All participants had genetically confirmed PWS. The TSH responses to thyroid-releasing hormone, free thyroxine (fT 4 ), and free triiodothyronine (fT 3 ) were measured in 21 subjects (14 females and 7 males, mean age 6.4 years). Capillary TT 4 was measured on neonatal screening samples from 23 subjects with PWS (14 females and 9 males), each of whom was matched for birth weight and sex with 4 anonymized controls.
Results: One subject with PWS had tertiary hypothyroidism. In the other 20 subjects, mean TSH level was 1.9 mU/l (range 0.8-4.2) at baseline and 21.8 mU/l (range 10.0-46.7) at 20 min (peak). Mean fT 4 concentration (10.4 pmol/l, range 8.2-13.5) was in the lower one-third of the normal range in 18 subjects, and mean fT 3 concentration (6.1 pmol/l, range 4.8-8.4) was above the median in 13 subjects. In neonates, mean TSH level was 3.1 mU/l (range 0.4-10.0) in subjects with PWS versus 3.3 mU/l (range 0.0-7.0) in controls, and mean TT 4 in subjects with PWS was 111% (range 17-203%) that of controls (p = n.s.).
Conclusion: Thyroid function was normal in the newborn subjects. In older children, frank hypothyroidism was found in only 1 of our 21 subjects.
The authors’ finding of lower fT 4 concentration in children and adolescents with PWS is in agreement with most studies. This could prompt some of us to introduce a thyroxine treatment. However, this seems to be unnecessary in almost all the children with PWS as demonstrated here, and thus levothyroxine treatment should not be routinely prescribed to youth with PWS. Indeed the same children had rather normal/high fT 3 concentrations. Increased thyroxine-to-triiodothyronine conversion also could contribute to the relative decrease in fT 4 in the presence of normal fT 3 concentrations. A possible explanation for this effect could be a leptin-mediated upregulation of type 2 deiodinase in response to the low-normal fT 4 levels. In addition, 18 of the 21 subjects of the study were receiving human growth hormone, which also could contribute to lower fT 4 concentrations through increased thyroxine-to-triiodothyronine conversion. Of note, the TRH test is not useful in our daily practice for the diagnosis of hypothyrodism and TRH is no more available widely.
Follow the thyroid after internal and external irradiation!

Twenty-Five Years after Chernobyl: Outcome of Radioiodine Treatment in Children and Adolescents with Very-High-Risk Radiation-Induced Differentiated Thyroid Carcinoma
Reiners C, Biko J, Haenscheid H, Hebestreit H, Kirinjuk S, Baranowski O, Marlowe RJ, Demidchik E, Drozd V, Demidchik Y
Department of Nuclear Medicine, University of Wurzburg, Wurzburg, Germany
J Clin Endocrinol Metab 2013 (E-pub ahead of print)
Context: After severe nuclear reactor accidents with release of radioactive iodine, elevated thyroid cancer risk in children and adolescents is considered one of the main health consequence for the population exposed.
Methods: The authors studied outcomes after 11.3 years’ median follow-up in a selected, very high-risk cohort, 234 Chernobyl-exposed Belarusian children and adolescents with thyroid carcinoma undergoing post-surgical radioiodine therapy (RIT) in Germany. Cumulatively 100 (134) children with (without) distant metastasis received a median 4 (2) RITs and 16.9 (6.6) GBq, corresponding to 368 (141) MBq/kg iodine-131. They evaluated outcomes by the response to therapy and disease status, mortality, and treatment toxicity.
Results: Of 229 patients evaluable for outcome, 147 (64.2%) attained complete remission (negative iodine-131 whole-body scan and TSH-stimulated serum Tg <1 µg/l), 69 (30.1%) showed nearly complete remission (complete response, except stimulated Tg 1-10 µg/l), and 11 (4.8%) had partial remission (Tg >10 µg/l, decrease from baseline in radioiodine uptake intensity in >/= 1 focus, in tumour volume, or in Tg). Except 2 recurrences (0.9%) after partial remission, no recurrences, progression, or disease-specific mortality were noted. One patient died of lung fibrosis 16 years post-therapy, 2 of apparently thyroid cancer-unrelated causes. The only RIT side effect observed was pulmonary fibrosis in 5/69 patients (7.2%) with disseminated lung metastases undergoing intensive pulmonary surveillance.
Conclusions: Even when radiation-induced differentiated thyroid carcinoma is advanced and initially suboptimally treated, response to subsequent radioiodine therapy and final outcomes are mostly favourable.
The population included in this cohort deserves some comments as they were severely affected and suboptimally treated: virtually all patients had papillary histology. At presentation, just under two-thirds had local invasion (stage pT 4 ); almost all had neck lymph node involvement. At final staging following post-RIT whole-body scintigraphy, somewhat under half the cohort had iodine-avid distant metastases, involving the lungs in all cases, plus the bone in 2 cases or the brain in 1. Lung metastases nearly always presented with disseminated ‘miliary’ spread (n = 54) or mixed diffuse-focal uptake (n = 38) rather than as clearly localized nodules (n = 8). A large majority of patients had undergone radical surgery, i.e. total thyroidectomy and lymph node dissection. Reoperation was necessary in under a quarter of the cohort. Fewer than a third of patients had received prior postoperative therapy besides thyroid hormone. Such therapy included iodine-131, typically in low cumulative activities (median 2.4 GBq), low-dose percutaneous irradiation (total 40 Gy) or single-agent or combination chemotherapy, mostly including bleomycin. Therefore the present observational study involved some of the highest-risk juveniles with Chernobyl radiation-induced differentiated thyroid carcinoma. The authors commented that due to the selection criteria for iodine-131 treatment in Germany, this cohort had frequent local invasion (64.1% pT 4 disease) and distant metastasis (42.7%) and nearly universal nodal involvement (97.0%). This is important to underline this as, if not, the conclusion of the article may appear overoptimistic.
With respect to the Fukushima nuclear incident, due to timely countermeasures (sheltering, evacuation, and a ban on potentially contaminated food and milk), the risk of radiation-induced differentiated thyroid carcinoma in children is much lower than it was after Chernobyl and – if there were any increased pediatric thyroid cancer incidence at all – it is very likely that due to early diagnosis by screening, advanced cases would be avoided.

Malignant and benign thyroid nodules after total body irradiation preceding hematopoietic cell transplantation during childhood
Vivanco M, Dalle JH, Alberti C, Lescoeur B, Yakouben K, Carel JC, Baruchel A, Leger J
Assistance Publique-Hopitaux de Paris, Service d'Endocrinologie Pediatrique, Centre de Reference des Maladies Endocriniennes Rares de la Croissance, Hopital Robert Debre, Paris, France
Eur J Endocrinol 2012;167:225-233
Background: Thyroid carcinoma after exposure to external radiation during childhood generally follows a linear dose-response relationship, but may plateau at the highest doses, and even slightly decrease at doses of more than 2,000 cGy. Age at irradiation, sex, and duration of follow-up have also been identified as determinants influencing the risk of thyroid tumors.The risk of radiation-induced benign thyroid nodules is also well known.
Methods: The authors aimed to determine the occurrence of thyroid nodules and carcinomas after fractionated total body irradiation (TBI) preceding hematopoietic stem cell transplantation (HSCT) for malignant hematological disease during childhood. They conducted a retrospective university hospital-based observational study. The participants were 76 patients receiving fractionated TBI between 1989 and 2009 as part of the conditioning regimen for HSCT to treat malignant hematological disease, with a median age of 8.2 (5.7-11.4) years, for whom the last ultrasound examination was performed at a median age of 14.

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