Background/Aims: A 33-year-old man was referred for the first time to the Division of Neurology because of the presence and progression of neurological symptoms. Dysphagia, weakness, reduced tear production, and nasal speech were present. In order to point the attention of late-onset triple A syndrome we describe this case and review the literature. Methods: Hormonal and biochemical evaluation, Schirmer test, tilt test and genetic testing for AAAS gene mutations. Results: Late-onset triple A syndrome caused by a novel homozygous missense mutation in the AAAS gene (A167V in exon 6) was diagnosed at least 17 years after symptom onset. Conclusions: The association between typical signs and symptoms of triple A syndrome should suggest the diagnosis even if they manifest in adulthood. The diagnosis should be confirmed by Schirmer test, endocrine testing (both basal and dynamic), genetic analysis, and detailed gastroenterological and neurological evaluations. Awareness of the possible late onset of the disease and of diagnosis in adulthood is still poor among clinicians, the acquaintance with the disease is more common among pediatricians. The importance of an adequate multidisciplinary clinical approach, dynamic testing for early diagnosis of adrenal insufficiency and periodical reassessment of adrenal function are emphasized. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Les glucocorticoïdes (GC), généralement sécrétés par le cortex surrénalien, exercent de très nombreuses fonctions dans l’organisme, via leur liaison au récepteur des glucocorticoïdes (GR). Les rares mutations inactivatrices du GR déjà décrites, sont responsables d’un syndrome de résistance aux GC et peuvent conduire à une hypertension artérielle (HTA), une hyperplasie surrénalienne (HBS), un hirsutisme et une obésité. Dans ce travail, nous avons caractérisé fonctionnellement 13 variants hétérozygotes du GR (expression, transactivation, localisation subcellulaire,...). Six variants du GR, découverts par séquençage à haut débit (NGS) ne sont pas pathogènes alors que 7 mutations hétérozygotes originales délétères ont été identifiées dans le cadre du protocole hospitalier de recherche clinique (Muta-GR). Ce PHRC a permis de préciser une prévalence à 5% de mutations inactivatrices du GR dans une cohorte de 100 patients avec HBS associée à une HTA et/ou un hypercortisolisme biologique sans signe clinique de Cushing.Une haploinsuffisance du GR, démontrée par la diminution d’induction par la déxamethasone du gène cible FKBP5, a été mise en évidence dans les fibroblastes cutanés de certains patients porteurs de mutations inactivatrices du GR. Ces patients présentent souvent un hypercorticisme avec hypokaliémie, aldostérone et rénine basse, signant un pseudohyperaldostéronisme. Nous avons démontré que le gène HSD11B2 codant pour l’enzyme 11β-HSD2, assurant l’inactivation des GC, est une cible directe du GR comme démontré par transfection transitoire de gène-rapporteur, RT-qPCR, LC/MSMS et ChIP. L’établissement des modèles de knock-in de mutations GR par stratégie Crispr/cas9 dans des lignées cellulaires préadipocytaires ou corticosurrénaliennes humaines s’est soldé par un échec. Ce travail devrait faciliter la sélection des patients chez qui la recherche de mutation inactivatrice du GR doit être faite et invite à un suivi régulier de ces patients. / Glucocorticoids (GC) regulate many essential biological functions by activating the glucocorticoid receptor (GR). GR loss-of function mutations are responsible for GC resistance syndrome, often associated with high blood pressure, hirsutism, bilateral adrenal hyperplasia (BAH) and obesity. Herein, functional characterization of 13 GR variants is presented (expression and binding studies, transactivation assays, subcellular localization) 6 variants were discovered with next-generating sequencing and had no functional impact on GR signaling while 7 GR loss-of-function mutations were mainly discovered during the National Clinical Hospital Research Program, Muta-GR. This PHRC discloses a 5% prevalence of GR loss-of-function mutations in a cohort of 100 patients with BAH, biological hypercortisolism and/or hypertension without Cushing signs. A GR haploinsuffisiency was demonstrated by a reduced dexamethasone-induced FKBP5 expression in skin fibroblasts of some patients harbouring GR loss-of-function mutations. These patients often presented with hypercorticism, hypokalemia, low renin and aldosterone levels, consistent with a pseudohypermineralocorticism. We showed that HSD11B2 encoding the 11β-HSD2 enzyme inactivating GC, is a direct GR target gene by transient transfection of reporter gene, RT-qPCR, LC/MSMS and ChIP. We failed to introduce GR loss-of-function mutations in human preadipocytes and adrenocortical cells by Crispr/Cas 9 technology. This work should facilitate selection of patients in whom GR mutation may be search, enabling an appropriate follow-up.

<p>  </p> <p>Glucocorticoids (GC) play a critical role in regulating the physiological response to stress. Disruptions to baseline levels due to stress can have negative implications on a variety of factors including growth and development, physical body conditions, metabolism, immune functions, and expression of normal behaviors, although this list is not exhaustive. When birds are unable to adapt to the stressor and return to homeostasis, the energy expenditure associated with the failed attempt at coping can lead to significant declines in the overall health, welfare, production, and performance of the bird. This can go on to impact producers and consumers as well, indicating the extensive repercussions of stress. Recently, scientists have been investigating thorough and efficient methods of quantifying stress in birds, such as measuring heterophil-to-lymphocyte ratio (HLR) or detecting glucocorticoid levels through enzyme-linked immunoassays (ELISA). However, the precise mechanism behind HLR increase during stress is unknown and ELISAs may not provide accurate results depending on when the blood is being measured. </p> <p><br></p> <p>GC are differentially released and exert their effects in a manner that is dependent on sex, age, and time. However, before investigating this, it was critical to validate the GC kits to ensure that they were measuring cortisol and corticosterone separately along with zero cross reactions with other precursors. Chapter 2 had 4 experiments carried out. The objective of experiment 1 was to validate ELISAs to ensure that they were measuring the GC accurately and separately since both cortisol and corticosterone were being measured. To do this, duck serum was pooled and charcoal-stripped to remove the presence of steroids. 3 standard curves were run to confirm that there was no cross reactivity. The objective of experiment 2 was to further validate the ELISA kits with mass spectrometry by checking for both glucocorticoids in the pooled samples. Once the validation process was complete, experiment 3 was carried out to look at the effect of ACTH stimulation on GC release. 16-week-old drakes and hens were given either intramuscular (IM) injections of cosyntropin (0.06 mg/kg) or saline as control. The cosyntropin dose was chosen according to previous studies reporting relatively high physiological responses, therefore, we wanted to replicate this. N was 10/sex/treatment. Blood was then collected at 0, 1, and 2 hours after injections and serum was analyzed by ELISAs. Lastly for experiment 4, 14-week-old developer drakes and hens at Maple Leaf Farms were assessed for a transportation stress experiment. Blood from 10 ducks/sex/time/barn were collected at 24 hours before transport to the breeder barn, immediately after a 1-hour transport, 24 hours after, and 1 week after transport. The results from experiment 1 yielded that both cortisol and corticosterone can be measured without the presence of unwanted contaminants or other products. Experiment 2 identified the greater sensitivity of mass spectrometry when reading GC levels, although the differences were linear. Experiment 3 showed that serum corticosterone levels were significantly increased at 1 hour after ACTH injections in both drakes and hens, with levels continuing to increase for the drakes. Serum cortisol levels were significantly increased at 1 hour after ACTH injections in both sexes, however, the hens had greater levels compared to the drakes. Serum cortisol levels returned to levels similar to that of saline-injected ducks at the 2-hour mark. Lastly, the transportation stress portion showed that cortisol was released at about 1/3 of corticosterone levels in both sexes. Hens showed increased levels of serum corticosterone compared to drakes at all time points except for 1 week after transport, and also had significantly increased serum cortisol levels at all time points. In conclusion, the ELISA kits were verified for future use when measuring GC as well as mass spectrometry. GC were detected in the ACTH and transportation stress experiments with hens displaying a greater sensitivity to GC release due to increased circulating levels compared to drakes. Although it was nonsignificant, there was a trend for GC to increase in response to transport. </p> <p><br></p> <p>There are sex differences in GC release and HLR for Pekin ducks and various challenges from the studies support this. With hens showing increased sensitivity to stress and drakes with more transient and gradual levels, we have consistently seen that both GC have differential roles in the stress response and not only is it critical to study both hormones, the timing of when measurements are taken are important as well to get a clear understanding of when the stress response is initiated. </p> <p><br></p> <p>Chapter 3 went further to understand the response of GC and HLR. The objective was to  investigate the release of cortisol and corticosterone in response to an ACTH dose response challenge. In Chapter 2, only one dose of cosyntropin was used and sample collection times only went to 2 hours after injections. In this study, 2 additional doses and an extra hour of sample collection were added to obtain more information. Pekin ducks were either given IM cosyntropin injections or saline for control, with an N of 10/sex/treatment. There were 3 treatment doses: High (0.06 mg/kg), medium (0.03 mg/kg), and low (0.015 mg/kg). All injections were given promptly at 0730 hours. Blood was collected at 0, 1, 2 and 3 hours after injections from the tibia veins to obtain serum for ELISAs. Blood smears were done to analyze HLR and sent to an independent lab to obtain values. The results indicated that both GC had significant sex x dose x time interactions. The low dose injection had no effect on corticosterone in hens with a slight increase for drakes at the first hour. The high dose for hens led to a spike in corticosterone levels at the first hour with a gradual decrease, and drakes had an increase that lasted for 2 hours until they returned to baseline at the last hour. The high dose in drakes stimulated cortisol release during the first 2 hours after injection with a similar effect in hens. However, hens had elevated levels compared to drakes. Finally, there was no dose response effect for HLR, although interestingly, the low dose injection elevated HLR even though there was no effect in GC. There were sex differences in the HLR response where the drakes given the high dose had levels that plateaued by the third hour, while the hens still had elevated levels. In conclusion, the ACTH dose-response test identified that ACTH has a dose-dependent effect in both GC and sex differences in their release. HLR also showed sex differences that did not depend on the dose given.</p> <p><br></p> <p>Chapter 4 observed acute exposure of GC in ducks. Pekin ducks were assigned 10/sex/treatment to receive either IM control, cortisol, or corticosterone injections. In addition, a low-dose cortisol treatment was given to represent the endogenous levels of cortisol compared to corticosterone. The control injections contained safflower oil, which was chosen as vehicle due to the low levels of genistein present. This is important as genistein is a plant estrogen and this could interact with the GC and alter the results. Blood was collected at 0, 1, 2, and 3 hours after injections for serum analysis with ELISAs, and blood smears were collected for complete blood count (CBC) differentials. Significant sex x treatment x time interactions were notable in both GC. Hens had significant increases at the first hour after injections in all treatments compared to controls, and drakes had increases at 2 hours after injections in all treatments except the low-dose cortisol. </p> <p><br></p> <p>After observing the effect of acute stress in ducks, the next step was to investigate the effects of chronic stress in chapter 5. Adult breeder Pekin ducks were randomly distributed into 3 groups: corticosterone, cortisol, or control treatments. The GC were in crystalline steroid form distributed through 2 capsules that were subcutaneously implanted on the backs of the neck. The ducks in the control group were given empty capsules. Blood smears, blood draws for serum, egg collection, body weights, and organ samples were collected over a period of 2 weeks. For the results, the corticosterone implants elevated corticosterone levels in both sexes. Interestingly, cortisol levels were elevated in both GC treatments in both sexes. Cortisol elevated HLR in drakes 1 day after implants with no effect from corticosterone. Hens had elevated HLR from both GC at all timepoints throughout the experiment. There were no significant differences in morphometrics in either sex. Corticosterone was not present in eggs, but cortisol was elevated in the albumen on day 7 and 14 of the experiment. Overall, there were sex differences in HLR where hens had greater levels in both GC treatments.</p>

La maladie de Cushing est caractérisée par une sécrétion excessive de l’hormone adrénocorticotrope (ACTH) à partir des tumeur corticotropes de l'hypophyse. Un excès d'ACTH entraîne un hypercortisolisme et provoque des symptômes tels que diabète, hypertension, obésité et les maladies cardiovasculaires entraînant un risque accru de mortalité si la maladie n’est pas traitée. Les tumeurs corticotropes sont caractérisées par la perte du rétro-contrôle négatif exercé par les glucocorticoïdes (GCs) sur la proopiomélanocortine (POMC) qui est le précurseur de l’ACTH : c'est la caractéristique majeure de la maladie de Cushing. Les causes de la résistance aux GC dans les adénomes corticotropes sont encore mal connues. Des études récentes ont montré une surexpression du récepteur du facteur de croissance épidermique (EGFR) dans les adénomes corticotropes provoquant une augmentation de l'activité du gène POMC et de la sécrétion d'ACTH. Les principaux objectifs de ce travail étaient de comprendre la relation entre la signalisation dérégulée de EGF et la résistance aux GCs. Dans le présent travail, nous avons identifié la voie JAK/STAT3 comme la principale voie de signalisation EGFR qui active la transcription du gène POMC. De plus, nous montrons que l'activation de la signalisation EGFR entraîne une résistance du promoteur POMC aux GCs et que l’activation de STAT3 est responsable de cette résistance. STAT3 affecte le mécanisme de transrepression de GR sans affecter le recrutement de GR au promoteur POMC. L’utilisation d’un inhibiteur de STAT3 restaure la répression de la transcription du promoteur POMC par les GCs. Nous avons aussi trouvé que 50% des adénomes corticotropes humains montrent une surexpression de la forme active de STAT3. Nous avons aussi étudié les mécanismes sous le contrôle des GCs qui régulent la prolifération cellulaire et qui pourraient être dérégulés dans la maladie de Cushing. CABLES1 est un régulateur négatif du cycle cellulaire et son expression est sous le contrôle des GCs. L’expression de CABLES1 est perdue dans 55 % des adénomes hypophysaires corticotropes, mais la cause de cette perte est encore mal comprise. Dans ce travail, nous avons identifié quatre variants faux-sens dans le gène CABLES1, deux chez de jeunes adultes (c.532G > A, c.718C > T) et deux chez des enfants (c.935G > A, et c.1388A > G) atteints de la maladie de Cushing. Les quatre variants touchent une région de la protéine CABLES1 qui est proche du motif de liaison de la kinase-3 dépendante des cyclines (CDK3). Ces variants ont perdu la capacité d’inhiber la croissance de cellules corticotropes tumorales (AtT20). Les quatre variantes sont donc des mutations de perte de fonction. En résumé, nos travaux révèlent le rôle important de STAT3 dans la résistance aux GC et ainsi, le blocage de l'action de STAT3 peut être une nouvelle stratégie pour le traitement de la maladie de Cushing. Nous avons aussi supporté un rôle de CABLES1 en tant que nouveau gène prédisposant aux tumeurs hypophysaires. / Cushing’s disease (CD) is characterized by excess secretion of adrenocorticotropic hormone (ACTH) from corticotroph tumors of the pituitary gland. Excessive ACTH leads to hypercortisolism that causes disabling symptoms such as diabetes, hypertension, obesity and cardiovascular disease resulting in an increased risk of mortality if it is not treated. Corticotroph tumors are characterized by the loss of glucocorticoid (GC) feedback repression of the proopiomelanocortin (POMC) that encodes the precursor of ACTH: this is the hallmark of CD. The causes of GC resistance in corticotroph adenomas of CD patients remain unknown. Recent findings showed overexpression of epidermal growth factor receptor (EGFR) in corticotroph adenomas causing increased POMC activity and ACTH secretion. The main objectives of this work were to understand the relationship between deregulated EGF signaling and GC resistance in the tumorigenesis of CD. In the present work, we identified the JAK/STAT3 pathway as the main EGFR pathway activating transcription of the POMC gene. We found that sustained activation of EGFR signaling or overactivation of STAT3 causes unresponsiveness of the POMC promoter to GCs and that activated STAT3 is responsible for GC resistance. STAT3 affects the transrepression mechanism of GR without affecting GR recruitment to the POMC promoter. The use of STAT3 inhibitor restores the repressive effect of GC on POMC transcription. Importantly, 50% of human corticotroph adenomas showed overexpression of activated STAT3. We also studied the mechanisms under the control of GCs that regulate cell proliferation and that could be deregulated in CD. CABLES1 is a negative cell cycle regulator, its expression is under the control of GC. CABLES1 expression is lost in 55 % of corticotroph adenomas and the underlying reasons remain unclear. In this work, we identified the presence of four missense variants in CABLES1 gene, two in young adults (c.532G > A, c.718C > T) and two in children (c.935G > A, and c.1388A > G) with CD. The four variants are close to the predicted cyclin-dependent kinase-3 (CDK3)-binding region of the CABLES1 protein. The variants have lost the ability to inhibit growth of corticotropinoma cells (AtT20). The four variants are thus loss of function mutations. In summary, our work revealed the important role of STAT3 in GC resistance and further indicates that inhibition of STAT3 action may be a novel strategy for CD treatment. We also provided evidence for a role of CABLES1 as a novel pituitary tumor-predisposing gene.

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No / Human aging involves a gradual decrease in cellular integrity that contributes to multiple complex disorders such as neurodegenerative disorders, cancer, diabetes, and cardiovascular diseases. Genome-wide association studies (GWAS) play a key role in discovering genetic variations that may contribute towards disease vulnerability. However, mostly disease-associated SNPs lie within non-coding part of the genome; majority of the variants are also present in linkage disequilibrium (LD) with the genome-wide significant SNPs (GWAS lead SNPs). Overall 600 SNPs were analyzed, out of which 291 returned RegulomeDB scores of 1-6. It was observed that just 4 out of those 291 SNPs show strong evidence of regulatory effects (RegulomeDB score < 3), while none of them includes any GWAS lead SNP. Nevertheless, this study demonstrates that by combining ENCODE project data along with GWAS reported information will provide important insights on the impact of a genetic variant-moving from GWAS towards understanding disease pathways. It is noteworthy that both genome-wide significant SNPs as well as the SNPs in LD must be considered for future studies; this may prove to be crucial in deciphering the potential regulatory elements involved in complex disorders and aging in particular.

Aktivator Protein-1 (AP-1) ist ein kollektiver Terminus für dimerische Transkriptionsfaktoren, die sich aus Fos- und Jun- Proteinen zusammensetzen. Diese Untereinheiten binden an eine gemeinsame, spezifische DNA-Sequenz, die AP-1 Bindungsstelle. Zusätzlich zu der gut dokumentierten Rolle des c-Fos Proteins in der Tumorgenese, wo dieses Gen als ein Aktivator beschrieben ist, übt AP-1 einen Einfluss auf mesenchymale Stromazellen und Immunzellen aus. Mesenchymale Knochenmarkszellen sind die Vorläuferzellen für Adipozyten, Osteoblasten, Chondrozyten, Myozyten und Fibroblasten. Die molekularen Mechanismen, welche die Differenzierungen regeln, sind noch weitgehend unerforscht. Der heterodimere Transkriptionsfaktor AP-1 übt eine wichtige Rolle in der Kontrolle der Zelldifferenzierung aus. Verschieden genetisch veränderte Mausmodelle untermauerten dies. Mäuse, welche das Fos-related antigen-1 (Fra1) oder eine kürzere Protein-Isoform von FosB (deltaFosB) überexpremieren, entwickelten, durch eine beschleunigte Differenzierung der Osteoblasten, eine Osteosklerose. Interessanterweise konnte gezeigt werden, dass die transgenen deltaFosB Mäuse weniger Fett haben. Die Stabilität und Aktivität von Fos Proteinen kann durch post-transkriptionale Modifizierungen geregelt werden. Basierend auf knockout Mausmodellen, wurde eine tragende Rolle für das wachstumsregulierende Enzym Rsk2 postuliert. Rsk2 spielt eine mögliche Rolle bei der Ausdifferenzierung von mesenchymalen Vorläuferzellen zu Osteoblasten und Adipozyten. Das Ziel dieser Arbeit war es molekulare Mechanismen zu finden, welche die unterschiedlichen Phänotypen (wild typ, fra1-tg, rsk2-defizient und fra1-tg/rsk2-defizient) charakterisieren. Die Knochenuntersuchungen der verschiedenen Genotypen zeigten, dass Fra1 und Rsk2, unabhängig voneinander, tragende Rollen im Knochenmetabolismus spielen. Quantitative Analysen von Adipozytenmarker, wie PPARgamma und C/EBPalpha zeigten, dass das Protein Fra1 die Adipozytenreifung in vivo und in vitro reguliert. Zusätzlich entwickelten die „doppel-mutierten“ fra1-tg/rsk2-/y Mäuse einen Lipodystrophy. Ein milderer Phänotyp wurde in den fra1-tg Tieren beobachtet, jedoch nicht in den Rsk2-knockout Mäusen. Zusätzlich wurde beobachtet, dass mesenchymale Zellen, welche Fra1 überexprimieren, gegen Glucocorticoid-induzierte Wachstumshemmung resistent waren. Diese Wirkung kann am wahrscheinlichsten durch die Fra1-vermittelte Suppression des Glucocorticoidrezeptors erklärt werden. Außerdem beeinflusste die Überexpression von Fra1 die Milzentwicklung. Leber und Herzanalysen zeigten, dass Fra1 kollagenhaltiges Gewebe induziert. Krankheiten wie Cholangitis und Fibrosen waren die Folge. / AP-1 transcription factor is a general name for multiple dimers formed by the association of Fos (or ATF) and Jun proteins. AP-1 acts as a sensor of changes in the cellular environment and thus, it is implicated in the modulation of cell proliferation, differentiation, transformation and cell death. Besides the well-documented role of c-Fos protein in oncogenesis, where this gene can function as a tumor promoter, AP-1 proteins are being recognized as regulators for mesenchymal stromal cell development and as regulators of immune cells. The mesenchymal stromal cells are the common progenitors for various mesenchymal lineages such as adipocytes, osteoblasts, chondrocytes, myocytes and fibroblasts. AP-1 seems to play a key role in the control of mesenchymal cell fate decision and differentiation. This is suggested by phenotypes of mice with a genetic modifications in either the Jun or the Fos component of AP-1. In particular, mice overexpressing the Fos-related antigen-1 (Fra1) or the short isoform of FosB (deltaFosB) have been found to develop osteosclerosis due to an accelerated differentiation of osteoblasts. Interestingly, mice overexpressing deltaFosB also developed less fat tissue. The activity of Fos proteins can be regulated by post-transcriptional modification. Based on knockout mouse model, a role for the growth factor regulated kinase Rsk2 was proposed in the differentiation of mesenchymal stromal cells to osteoblasts as well as in fat tissue development. Goal in this study was to identify the molecular mechanisms explaining the differences between the wild type, fra1-tg, rsk2-deficient and fra1-tg/rsk2-deficient phenotypes. The comparison of the bones of the different mice genotypes revealed, that Fra1 and Rsk2 were independently regulating bone metabolism. Quantitative analysis of adipocyte markers expressions, like PPARgamma and C/EBPalpha revealed, that Fra1 overexpression was blocking adipocyte maturation in vivo and in vitro. Moreover, the in vivo results show that the fra1-tg/rsk2-/y mice develop a severe lipodystrophy. A milder phenotype was observed in the parental fra1-tg strain but not in the Rsk2 knockout strain. Additionally, it was been observed, that mesenchymal cells overexpressing Fra1 were resistant to glucocorticoid-induced growth inhibition. This effect can most likely be explained by Fra1-mediated downregulation of the glucocorticoid receptor. Furthermore, Fra1 overexpression influenced spleen development. Liver and heart analyses showed that Fra1 overexpression induced collagen tissue. Diseases like cholangitis and fibrosis were the outcome.