bluuebunny/crossref_metadata_2025 huggingface dataset:
Comprehensive and Cost-Effective Strategy for Genetic Screening of Congenital Adrenal Hyperplasia (CAH) in India
Authors: Ravichandran Lavanya , Korula Sophy , Asha H S , Varghese Deny , Parthiban R , Jabasteen J , Jebasingh Felix , Kapoor Nitin , Cherian Kripa Elizabeth , Mathai Sarah , Simon Anna , Paul Thomas V , Thomas Nihal , Chapla Aaron
Published: May 2021

Background: With substantial challenges in molecular analysis of 21 hydroxylase deficiency and lack of studies in extended panel of genes implicated in CAH, genetic diagnosis is largely unavailable and unaffordable in India. Therefore, we aim to develop a cost-effective screening strategy in CAH using Allele Specific PCR and Targeted Next-Generation Sequencing (NGS). Methods: Long range PCR and restriction digestion were utilized to specifically amplify the CYP21A2 gene whereas multiplex PCR was used to amplify CYP11B1, CYP17A1, CYP19A1 and POR genes. In house developed Allele Specific PCR (ASPCR) for 8 hotspot mutations in CYP21A2 gene and targeted NGS for five genes was carried out. The results were validated using Sanger sequencing and MLPA. Results: Of the 50 patients suspected for 21 hydroxylase deficiency, 64% (n=32) were of Salt Wasting phenotype (SW), 30% (n=15) with Simple Virilizing (SV) phenotype and 6% (n=3) of the study population were suspected for non-classical (NC) CAH. The mutation positive rate of ASPCR was 86% (n=43). Seven patients carried more than two biallelic mutations indicating smaller gene conversions. The predominant mutation identified among the study subjects was I2G splice variant in SW phenotype (38%) and I172N in the SV phenotype (41%). Based on the Long range PCR amplification and restriction digestion we identified one patient with large gene conversion and one patient with large 30kb deletion. These results were confirmed with MLPA.

Additionally, utilizing the Targeted NGS we identified five patients with CYP21A2 variants (two patients with novel variants c.1274G>T, c.17_18delTG and two other variants in three patients - c.1451G>C, c.143A>G). We also identified a CYP19A1:c.1142A>T gene variant in a patient who was initially suspected for 21 hydroxylase deficiency. Out of six patients with 11 beta hydroxylase deficiency four patients were positive for homozygous CYP11B1 variants (c.1201-1G>A, c.1200 + 1delG, c.412C>T) and two patients with compound heterozygous variants (c.1024C>T and c.1012dupC, c.623G>A and c.412C>T).

Discussion: Utilizing the novel allele specific PCR followed by NGS we identified a total of 96% (48/50) of 21 hydroxylase deficiency patients with homozygous or compound heterozygous mutations and 2% (2/50) were positive for single heterozygous variant in CYP21A2 gene. ASPCR followed by multigene targeted NGS assay for genetic screening in CAH has shown to be a sensitive and specific strategy established in a clinical setting. To best of our knowledge this is the most cost-effective and comprehensive multigene screening carried out in India.
Heterodimerization and Subcellular Distribution of Melatonin and Cannabinoid Type 1 Receptors
Authors: Cui Yuanxu , Jockers Ralf , Lahuna Olivier
Published: May 2021

Membrane receptors belonging to the G proteins coupled receptors (GPCRs) form the largest family of proteins in the human genome with more than 800 members. Until recently GPCRs functions were thought to occur only at the plasma membrane after activation upon binding of their cognate ligand. However evidences show that many functional GPCRs are found in intracellular compartments opening new direction of research to understand their roles in a cellular context. Among these intracellular compartments mitochondria are the latest organelle in which some GPCRs were identified. Melatonin receptor type 1 (MT1) and cannabinoid receptor type 1 (CB1) were identified in mouse neuronal mitochondria where they were shown to exert an inhibitory action on cytochrome c release (MT1) or on the respiratory chain (CB1). Using several techniques my current results describe a new cross-talk between MT1 and CB1 receptors. Confocal analysis of immunofluorescence experiments of cells coexpressing both receptors showed a high degree of colocalisation. A combination of coimmunoprecipitation experiments performed on extracts of transfected HEK293T or HeLa cell lines and immunodetection of receptors by Western-blot revealed that MT1 and CB1 receptors can physically interact to form heterodimers in absence of ligand. Heterodimers formation was also confirmed by Proximity Ligation Assay (PLA) experiments in live HEK293T and HeLa cells. Confocal analysis revealed a colocalisation of PLA staining with the mitochondrial marker TOMM20. Experiments in relation with the functional role of MT1 and CB1 in mitochondria are ongoing.
Single Nucleus RNA-Sequencing Reveals Overall Conservation of Hypothalamic Cell Identities but Differential Expression of Specific Genes in the Magel2 Null Mouse Model of Prader-Willi Syndrome
Authors: Yu Hui , Low Malcolm James
Published: May 2021

Prader-Willi syndrome (PWS) is a genetic disorder affecting 1 in 10,000 to 30,000 live births. Diagnostic features of PWS including insatiable appetite and obesity are well-defined and many are associated with disruption of hypothalamic function. PWS is caused by sporadic or inherited loss of expression from the paternal allele of one or more maternally imprinted/silenced genes located in chromosomal region 15q11-q13 that encompasses five protein coding genes Mkrn3, Magel2, Necdin and Snurf-Snprn and a family of snoRNAs. Seminal studies have indicated that isolated Magel2 gene silencing plays a pivotal role in the development of many, but not all, clinical features of PWS. Magel2 is highly expressed in the hypothalamus and loss of function studies revealed substantial cellular and molecular changes in hypothalamic neurons located in the suprachiasmatic, paraventricular, supraoptic and arcuate nuclei and the lateral hypothalamus. In addition to neuronal alterations, loss of MAGEL2 increases the density and activation of microglia in adult hypothalamus. In the current study, we characterized global changes in hypothalamic gene expression and searched for novel cell populations associated with loss of Magel2 expression using single nucleus RNA sequencing. Single cell nuclei were isolated in two technical replicates per group from hypothalami of adult male and female Magel2-null (C57BL/6-Magel2tm1Stw/J) and wild type sibling mice for the 10X genomics scRNA-seq pipeline. A total of 63,470 cells divided approximately equally by sex and Magel2 genotype were analyzed. Unsupervised cell clustering identified 19 distinct clusters in males (10 neuronal and 9 non-neuronal) and 21 clusters in females (11 neuronal and 10 non-neuronal) based on their transcriptional profiles of signature genes. The percentages of total cells and the transcriptional profiles of each defined cluster from all four combinations of genotype and sex were nearly identical, indicating that loss-of-function of Magel2 does not alter overall cell cluster identities in the hypothalamus. However, a quantitative analysis of gene expression profiles from all individual clusters demonstrated upregulation of a set of genes including Fkbp5, Zbtb16, Htra1, 2900097C17Rik and 1700030F04Rik predominantly in oligodendrocytes and astrocytes in both sexes of Magel2 null mice. In contrast, the majority of down-regulated genes were found in neuronal cell clusters in both sexes of Magel2 null mice. Our current study is the first to characterize cellular and genetic changes in the whole hypothalamus due to the lack of MAGEL2. The data provide a valuable resource for elucidating the regulatory mechanisms of MAGEL2 for the pathogenesis of PWS and shed light on the discovery of new candidate targets for the potential treatment of PWS. This study is supported by the Foundation for Prader-Willi Research and NIH grant R01DK068400.
Oral Octreotide Capsules Lowered Incidence and Improved Severity of Acromegaly Symptoms Compared to Injectable Somatostatin Receptor Ligands—Results From the MPOWERED Trial
Authors: Biermasz Nienke , Fleseriu Maria , Dreval Akexander V , Pokramovich Yulia , Bondar Irina , Isaeva Elena , Molitch Mark E , Macut Djuro P , Leonova Nina , Raverot Gerald , Gilgun-Sherki Yossi , Ludlam William H , Patou Gary , Haviv Asi , Gordon Murray B , Urbanavicius Vaidotas , Knispelis Robertas , Melmed Shlomo , Strasburger Christian J
Published: May 2021

Background: Patients with acromegaly may have high symptom burden. The phase 3 MPOWERED trial assessed control of acromegaly by oral octreotide capsules (OOC; MYCAPSSA®) in comparison to injectable somatostatin receptor ligands (iSRLs) in patients responding to both OOC and iSRLs. iSRLs have been first-line medical treatment for patients with acromegaly for decades. OOC are newly approved in the US for patients previously controlled on iSRLs.

Methods: Eligibility criteria for MPOWERED included acromegaly diagnosis, biochemical control of acromegaly (insulin-like growth factor I <1.3 × upper limit of normal; mean integrated growth hormone, <2.5 ng/mL) and ≥6 months’ iSRL (octreotide, lanreotide) treatment. Eligible patients entered a 26-week Run-in phase to determine the effective OOC dose; responders at week 24 then entered a 36-week randomized controlled treatment (RCT) phase receiving OOC or iSRLs. Acromegaly symptom number and severity (mild to severe, 1-3) were collected. Total score was calculated by summating all severity scores (Acromegaly Index of Severity [AIS]). Symptom results were assessed using total AIS score and proportion of patients experiencing individual symptoms.

Results: At beginning of Run-in, average AIS score of 92 randomized patients was 4.52, representative of symptoms experienced while previously receiving iSRLs. After 26 weeks’ OOC treatment at end of Run-in, average AIS score was significantly reduced to 3.46 (P<0.001). More than 80% of patients on OOC improved or maintained AIS score during Run-in compared to baseline. Over this 26-week period, there was a significant reduction in extremity swelling (P=0.01) and fatigue (P=0.03). During the RCT, of patients randomized to OOC (n=55), 73% maintained or improved AIS score, and 75% maintained or reduced overall number of active symptoms. In comparison, 68% of those randomized to iSRLs (n=37) maintained or improved AIS score, and 70% maintained or reduced overall number of active symptoms.

Conclusion: Results from MPOWERED show that patients receiving OOC had significant improvement in number and severity of acromegaly symptoms after switching from iSRLs. These findings validate previous results from a phase 3 study of OOC in acromegaly in which patients switching to OOC from iSRLs showed significant reduction in joint pain, extremity swelling, and fatigue.1

1Melmed S, et al. JCEM. 2015;100(4):1699-1708.
SIX3 Variant Causes Pituitary Stalk Interruption Syndrome and Combined Pituitary Hormone Deficiency
Authors: Bando Hironori , Brinkmeier Michelle , Gergics Peter , Fang Qing , Mortensen Amanda Helen , Ozel Ayse Bilge , Ma Qianyi , Li Jun , Reynaud Rachel , Castinetti Frederic , Brue Thierry Christian , Camper Sally Ann
Published: May 2021

The genetic basis for congenital hypopituitarism and related disorders is beginning to emerge, and over 30 causal genes have been identified. Mutations in some of these genes can also cause holoprosencephaly (HPE) or septo-optic dysplasia. SIX3 is a homeodomain protein expressed in the developing brain, pituitary gland, and eye. Heterozygous mutations in SIX3 cause variable HPE in humans and mice. We identified two children with neonatal GH and TSH deficiency and stalk interruption who were doubly heterozygous for rare, likely deleterious variants in SIX3 and POU1F1. Functional studies demonstrated that both variants are disruptive. We used Six3 and Pou1f1 loss of function mice to assess the genetic interaction between Six3 and Pou1f1. Six3 heterozygotes have variable pituitary gland dysmorphology, while Pou1f1 heterozygotes are normal. A significant portion of the Six3+/-; Pou1f1+/dw doubly heterozygous mice have a more pronounced pituitary phenotype than Six3+/-, supporting the possibility of digenic pituitary disease. To understand the role of SIX3 in pituitary and hypothalamic development, we used Prop1-cre and Nkx2.1-cre to delete Six3. Disruption of Six3 expression in Rathke’s pouch caused poor activation of Lhx3 expression and arrested anterior pituitary development. The Nkx2.1-cre, Six3flox/flox embryos had no evidence of infundibulum evagination and failed to induce FGF and BMP signaling, which normally drive expansion of Rathke’s pouch. By E11.5 cells in Rathke’s pouch underwent apoptosis. The Nkx2.1-cre, Six3flox/flox embryos failed to activate expression of Lhx2 and Tbx3 in the neural ectoderm. These embryos had elevated CCND1, MYCN, and Axin2 expression in the area of the presumptive infundibulum. This indicates that SIX3 is necessary to repress cell proliferation and Wnt/beta-catenin signals to promote formation of the pituitary stalk. Thus, Six3 has essential roles in both the neural and oral ectoderm for hypothalamic and pituitary development, respectively. Heterozygous loss of function variants in SIX3 could be a contributor to multiple pituitary hormone deficiencies in children, especially if there are associated craniofacial abnormalities or PSIS.
A Mouse Model of Shiftwork Reveals Sex-Specific Impairments in Circadian Behavior and Reproductive Tissue Timekeeping
Authors: Yaw Alexandra M , Mclane-Svoboda Autumn , Nguyen Duong , Duong Thu Van Quynh , Hoffmann Hanne Mette
Published: May 2021

Approximately 21 million Americans are employed in nighttime or rotating shiftwork, which is linked with numerous health risks, including reduced reproductive success. While there is a strong body of clinical evidence associating shift workers with deficits in fertility, very little is known about the mechanisms by which shiftwork produces reproductive dysfunction. One important feature of shiftwork is extensive exposure to light at night. Light is one of the strongest timing cues for the circadian system, where exposure to mistimed light (light at night) can disrupt timekeeping throughout the body, leading to a mistiming of physiological processes and resulting in disease development. Our goal is to determine if a light-based model of rotating shiftwork (shift-light), composed of an alternating 6 hour phase advance or delay every 4 days for 5-10 weeks, disrupts circadian rhythms in the reproductive hypothalamic-pituitary-gonadal (HPG) axis, leading to mis-timed tissue rhythms and reduced fertility. We hypothesize that shift-light disrupts wheel running behavior and underlying cellular circadian rhythms in the HPG axis, resulting in reduced reproductive function. Using the validated circadian Per2:luciferase reporter mice, we assessed wheel-running behavior and Per2:luciferase rhythms in tissue explants from males and females. Behavioral data revealed that both sexes adapt their wheel running to shift-light paradigm; however, females, but not males, displayed a significant deficit in their ability to entrain to phase advances following 4 shifts. This sex-specific disruption was supported by preliminary tissue explant Per2:luciferase rhythms, which suggest that shift-light alters tissue level circadian phase synchrony in female, but not male, HPG axis tissues. Importantly, females exhibited shortened estrous cycling during shift-light, suggesting the altered HPG axis synchrony could be directly impacting reproductive function. We are currently working to extend this work to determine how this desynchrony impacts hormone release, including luteinizing hormone and follicle stimulating hormone. Together, this work provides insight into how shiftwork may influence circadian rhythms in reproductive tissues and suggests that females may have increased vulnerability to reproductive deficits from shiftwork.
Canine Pituitary Organoids as 3D In Vitro Model for Cushing Disease
Authors: Sanders Karin , Ringnalda Femke C A , van de Wetering Marc L , Kooistra Hans S , Meij Björn P , Clevers Hans , Galac Sara
Published: May 2021

Cushing disease (CD) is a serious endocrine disorder that is most often caused by an ACTH-secreting pituitary adenoma. Patients can be treated medically when surgery is not an option or was unsuccessful. However, currently used pituitary-targeting drugs are effective in only 40% of patients. To efficiently identify new pituitary-targeting treatment options, we need an in vitro system that closely mimics in vivo conditions. We therefore aimed to establish organoid cultures of normal anterior pituitary and corticotroph adenomas. Organoids or tumoroids are miniature three-dimensional (3D) structures grown from stem cells, that closely resemble the organ or tumor they originate from. Because CD is a thousand times more prevalent in dogs than in humans, and hypophysectomy is the treatment of choice, we used canine tissues. Normal anterior pituitary glands were collected from three healthy dogs that were euthanized for reasons unrelated to the current study. Corticotroph adenomas were collected from six dogs that underwent transsphenoidal hypophysectomy at our University Clinic. The dogs were diagnosed with CD based on clinical signs, endocrine testing, and CT scan imaging. Normal anterior pituitary and corticotroph adenoma cells were cultured in a 3D matrix (basement membrane extract) with anterior pituitary organoid medium containing specific growth factors and ligands, which was refreshed twice a week. The organoids and tumoroids were characterized with histopathology and RT-qPCR. Structures resembling organoids or tumoroids grew from all nine samples (3 normal, 6 adenoma) that were put in culture. Both cystic and dense structures were observed. The organoids and tumoroids expanded rapidly, and could be passaged once every week. The organoids and tumoroids were successfully cultured up until passage number 10, and were then frozen down. Histopathology showed that the organoid or tumoroid cells morphologically resembled healthy anterior pituitary or corticotroph adenoma cells. All organoids cultures expressed mRNA of pituitary stem cell markers SOX2 and SOX9. This study shows that corticotroph adenomas can be cultured as tumoroids in vitro, something not previously published in any species. Based on the many opportunities in organoid culture (e.g., high-throughput drug screenings, gene editing, studying developmental processes), we expect that this in vitro model will pave the way to efficiently and reliably identify new treatment options for CD. Not only for humans, but also for our best friends: dogs.
Development of a Novel Tetracycline-Inducible Kiss1-Cre Mouse Line for Temporally Controlled Gene Deletion in Kisspeptin Neurons
Authors: Negron Ariel L , Levine Jon E , Radovick Sally
Published: May 2021

The tetracycline (Tet)-controlled inducible system is the most widely used reversible system for transgenic expression in mice. Previously, we generated a GnRH-CreTeR mouse model, using a first-generation Tet-inducible system to temporally induce expression of Cre recombinase in GnRH neurons. Recently, the Tet-inducible system has undergone several modifications to significantly reduce previous limitations that include leaky background expression and lower sensitivity to tetracycline induction. Therefore, we have developed a novel mouse model bearing a Tet-inducible kisspeptin-Cre allele (iKiss-Cre mouse) that will enable temporal control over the selective deletion of genes from Kiss1 neurons. This temporally controlled gene deletion will eliminate a longstanding technical limitation of conventional steroid receptor knockout models in which steroid regulation of the axis is confounded by steroid developmental and organizational effects in the reproductive axis. Two mouse lines were generated. The first line targets kisspeptin neurons with a third generation Tet-inducible reverse tetracycline transactivator (rtTA, Tet-On 3G) expressed under the control of the Kiss1 allele. Using CRISPR-Cas9 technology, we inserted a cassette containing an internal ribosome entry site (IRES) sequence followed by the rtTA downstream of the Kiss1 coding region as was previously done using Cre recombinase. Transcription of the recombinant Kiss1 allele yields a bicistronic messenger RNA, from which both kisspeptin and rtTA are independently translated. The second mouse line, TRE-Cre mice, was constructed to express Cre recombinase under control of the PTRE3G promoter. The PTRE3G promoter is bidirectional to allow simultaneous monitoring of Cre expression and a second florescent protein reporter (ZsGreen1). The Kiss1-rtTA only binds to and activates the PTRE3G promoter in the presence of doxycycline (an analog of tetracycline). Offspring from breeding of these mouse lines, iKiss-Cre, result in a system capable of generating Tet-induced expression of Cre recombinase in kisspeptin neurons. In order to document specificity and sensitivity of this system, we performed immunofluorescent staining and observed colocalization of kisspeptin with Cre recombinase in brain sections of iKiss-Cre mice only after doxycycline treatment. We will use this model to investigate negative feedback actions of E2 on the adultpulse generator after E2 exerts its organizational actions on maturing kisspeptin neurons. We expect that this mouse model will become a major tool used by the neuroendocrine community and serve as proof of principle for development of similar inducible knockout models employing the current generation of inducible methodologies.
MIF Inhibition Suppresses Cell Viability and Induces Apoptosis via the ATF4-CHOP Pathway in Mouse Pituitary AtT-20 Cells
Authors: Wang Jason , Satou Motoyasu , Tateno Tae , Willette Sarah , Yang Rui Zhe , Kitayoshi Fumika , Teramachi Mariko , Sugimoto Hiroyuki , Chik Constance L , Tateno Toru
Published: May 2021

Cushing’s disease (CD) is characterized by cortisol overproduction due to ACTH hypersecretion from a pituitary tumour (PT). With an incidence of approximately 1.2 to 2.4 cases per million per year, CD patients have higher rates of morbidity and mortality than the general population. Surgical management is currently the first therapeutic option. However, remission rates vary between studies, and patients may suffer from complications caused by hormonal abnormalities from remnant PT tissues, the surgery itself, as medical treatment options are limited. Macrophage migratory inhibitory factor (MIF) is a cytokine expressed in various tumors, including ACTH-producing PTs, and has been found to play a crucial role in tumorigenesis. Previous studies demonstrate that MIF regulates cell growth via the signal transducer and activator of transcription 3 (STAT3) pathway, the mammalian target of rapamycin (mTOR) pathway, and autophagy. Together, these indicate MIF as a potential therapeutic target for PTs. However, the role of MIF in ACTH-producing PTs remains unknown. Using mouse ACTH-producing PT cells, AtT-20 cells as a model, we established that MIF overexpression led to increased cell growth. In contrast, pharmacological MIF inhibition by 4-iodo-6-phenylpyrimidine (4-IPP) and (S,R)-3-(4-Hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid (ISO-1) and genetic MIF downregulation by siRNA both suppressed cell viability and induced apoptosis, suggesting an anti-apoptotic role of MIF. Genetic MIF downregulation also increased the expression of apoptosis-inducible genes such as activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), and reduced ACTH production. However, pharmacological MIF inhibition had no effect on ACTH production, which suggests that the mechanism of pharmacological MIF inhibition may be different from MIF downregulation. Neither MIF upregulation nor downregulation affected cell signalling pathways such as the STAT3 pathway, the mTOR pathway, or autophagy. Our findings suggest that MIF inhibition can be a viable therapeutic approach for ACTH-producing PTs.
ScRNA-seq Reveals a Role of Mammary Luminal Epithelium in Adipocyte Adaptations
Authors: Santos Luis , Arneson Douglas , Chella Krishnan Karthickeyan , Ahn In Sook , Diamante Graciel , Cely Ingrid , Butte Atul , Lusis Aldons , Yang Xia , Rajbhandari Prashant
Published: May 2021

Almost four decades of research suggest a dynamic role of ductal epithelial cells in adipocyte adaptation in mammary gland white adipose tissue (mgWAT), but factors that mediate such communication are not known. Here, we identify a complex intercellular crosstalk in mgWAT revealed by single-cell RNA-seq (scRNA-seq) and comprehensive data analysis suggest that epithelial luminal cells during cold exposure undergo major transcriptomic changes that lead to the expression of an array of genes that encode for secreted factors involved in adipose metabolism such as Adropin (Enho), neuregulin 4 (Nrg4), angiopoietin-like 4 (Angptl4), lipocalin 2 (Lcn2), milk fat globule-EGF factor 8 (Mfge8), Insulin-like growth factor-binding protein 1 (Igfbp1), and haptoglobin (Hp). To define the mammary epithelial secretome, we coin the phrase “mammokines”. We validated our cluster annotations and cluster-specific transcriptomics using eight different adipose scRNA-seq datasets including Tabula Muris and Tabula Muris Senis. In situ mRNA hybridization and ex vivo isolated mgWAT luminal cells show highly localized expression of mammokines in mammary ducts. Trajectory inference demonstrates that cold-exposed luminal cells have similar transcriptional profiles to lactation post-involution (PI), a phase defined by reappearance and maintenance of adipocytes in the mammary gland. Concomitantly, we found that under cold exposure female mgWAT maintains more adipogenic and less thermogenic potential than male scWAT and ex vivo removal of luminal epithelial cells from mgWAT markedly potentiates beige adipocyte differentiation. Conditioned media from isolated mammary epithelial cells treated with isoproterenol suppressed thermogenesis in differentiated beige/brown adipocytes and treatment of beige/brown differentiated adipocyte with mammokine LCN2 suppresses thermogenesis and increases adipogenesis. Finally, we find that mice lacking LCN2 show markedly higher cold-dependent thermogenesis in mgWAT than controls, and reconstitution of LCN2 in the mgWAT of LCN2 knockout mice promotes inhibition of thermogenesis. These results show a previously unknown role of mammary epithelium in adipocyte metabolism and suggest a potentially redundant evolutionary role of mammokines in maintaining mgWAT adiposity during cold exposure. Our data highlight mammary gland epithelium as a highly active metabolic cell type and mammokines could have broader implications in mammary gland physiology and lipid metabolism.
Recessive PRDM13 Mutations Result in Hypogonadotropic Hypogonadism and Cerebellar Hypoplasia
Authors: Oleari Roberto , Whittaker Danielle , Gregory Louise Cheryl , Albert Basson , Cariboni Anna Maria , Dattani Mehul Tulsidas
Published: May 2021

PRDM13 (PR Domain containing 13) is a putative chromatin modifier and transcriptional regulator that functions downstream of the transcription factor PTF1A. Here, we report a novel, recessive syndrome associated with PRDM13 mutation. Patients exhibited intellectual disability, ataxia with cerebellar hypoplasia, scoliosis and delayed puberty with hypogonadotropic hypogonadism (HH). We investigated the development of hypothalamic neurons and the cerebellum in mice homozygous for a Prdm13 mutant allele. Cerebellar hypoplasia was evident, but male gonadal development appeared unaffected in these mutants. As PTF1A has been linked to early GABAergic neuronal cell fate regulation in the spinal cord, we examined GABAergic neuron progenitor development in the hypothalamus and cerebellum. A significant reduction in the number of Kisspeptin neurons in the hypothalamus and PAX2+ progenitors emerging from the cerebellar ventricular zone was observed. The latter was accompanied by ectopic expression of the glutamatergic lineage marker TLX3. Together, these findings identify PRDM13 as a critical regulator of GABAergic cell fate during neurodevelopment, providing a mechanistic explanation for the co-occurrence of HH and cerebellar hypoplasia in this syndrome. To our knowledge, this is the first evidence linking disrupted regulation of Kiss1 neurons to CHH in humans.
Spatial Transcriptomics for the Analysis of Human Pituitary Development
Authors: Matsumoto Ryusaku , Kabata Mio , Suga Hidetaka , Yamamoto Takuya
Published: May 2021

The pituitary develops from oral ectoderm in contact with the adjacent hypothalamus. However, the precise mechanisms underlying pituitary development in concert with plural tissues are not fully understood, especially in human. A protocol to induce pituitary cells from human induced pluripotent stem cells (hiPSCs) has been established and applied to study pituitary development and disorders. In the method, oral ectoderm and hypothalamus are induced in one organoid, which enables recapitulation of the interactions between these tissues during embryonic development. It leads to self-organization of pituitary cells. Recently, spatial transcriptome technology has been developed and is suitable for the analysis of tissue interactions. Here, we utilized spatial transcriptomics to analyze pituitary organoids, especially focusing on the mechanisms regulating pituitary progenitor cell differentiation. Spatial transcriptomics revealed that the organoids consisted of several cell populations including hypothalamus, oral ectoderm, neural retina, and cortex neuron cells. Pituitary progenitor cells, characterized by the upregulation of LHX3, were included as part of the oral ectoderm population. Further analysis of the population identified human pituitary progenitor-specific genes including many causal genes for congenital hypopituitarism (CPH). Finally, using spatially resolved gene expression data, we examined the hypothalamic population that was in contact with pituitary progenitor cells and identified hypothalamic factors that might regulate progenitor cell differentiation in a paracrine manner. The genes upregulated in the pituitary progenitor and neighboring hypothalamus cell populations are potential causal gene candidates for CPH. In conclusion, spatial transcriptomics provides a novel platform to analyze tissue interaction networks during human pituitary development.
A Case of Inappropriate Antidiuretic Hormone Secretion Syndrome Associated With COVID-19 Pneumonia Treated With Boluses of Hypertonic Saline for Reversal of Symptomatic Hyponatremia
Authors: Miranda Gonzalo Francisco , Rodríguez Karen Evelyn Ramos , Research Clinical
Published: May 2021

Hyponatremia is the most common electrolyte disorder, which can occur in outpatients and hospitalized patients, so both first-contact doctors and specialists must keep up-to-date on the prevention, recognition, diagnosis and management of this complication.

A 68-year-old male patient presents to Dos de Mayo National Hospital Emergency Department. He was diagnosed as COVID-19 pneumonia and hospitalized for management of acute respiratory failure. The patient had neurological impairment associated with poor oral tolerance. Initial laboratory examinations were C-reactive protein in 363.5 mg/L, serum sodium of 128.42 mmol/L and urine sodium was 83 meq/L. Osmolality in plasma was 266.15 mOsm/Kg and urine osmolality was 420 mOsm/Kg. Thyroid function tests as well as cortisol levels were in normal range. Our patient was diagnosed as SIAD by hyponatremia, osmolality in plasma <275 mOsm / kg, urine osmolality > 100 mOsm / kg, urine sodium > 40 mEq / l, euvolemic state and exclusion of cortisol and thyroid hormone deficiency. Treatment of hyponatremia was initiated and rapidly elevate plasma sodium by 4 meq/l in the first 6 hours. There was clinical improvement. Blood sodium levels ranged from 115 to 135 mmol/L with bolus therapy of hypertonic solutions in 72 hours. Intravenous boluses of hypertonic saline should be administered to rapidly elevate plasma sodium by 4 to 6 mEq/L in the first 6 hours. The data shows that fluid bolus therapy is more effective in acutely elevating plasma sodium than traditional low-dose hypertonic saline infusion that may lead to avoidable deaths according to recent guidelines. In this case a strategy based with bolus therapy for reversal of hyponatremia was used effectively.

A number of cases of COVID-19 pneumonia are associated with SIAD. The presence of SIAD could be a clue to diagnosing COVID-19. SIAD is a major complication of COVID-19 and could be the first and only manifestation. In cases of SIAD without a clear etiology we should suspect COVID-19 in a patient with respiratory distress in the current pandemic.

Syndrome of inappropiate antidiuresis (SIAD) should be assessed in every patient with COVID-19 as their treatment and early identification decreases mortality. The association between COVID-19 pneumonia and SIAD should be further identified, requiring doctors to be aware of this condition. Additional studies are required to determine the incidence and pathogenesis of SIAD in patients with COVID-19.
Acknowledging the Blind Spot of Bitemporal Hemianopsia: Keeping a High Index of Suspicion for Pituitary Macroadenomas in Uncommon Presentations
Authors: Raco Joseph , Macias Maria , Jain Rohit
Published: May 2021

Background: Hyperglycemia in patients with type 2 diabetes mellitus commonly manifests as symptoms of polyuria, polydipsia, fatigue, and weight loss as a result of insulin resistance. In cases of severe hyperglycemia, patients may also experience visual disturbances and dizziness as a result of swelling of the lens and dehydration respectively. These symptoms are not generally accompanied by gait disturbance or fixed, focal deficits on neurological examination. As such, symptoms such as double vision, peripheral field vision loss, cranial nerve deficits, or significant unsteadiness may warrant a more extensive neurologic workup rather than simply attributing all symptoms to hyperglycemia. Clinical Case: A 57-year-old woman presented to the emergency department with five days of fatigue, polyuria, polydipsia and pre-syncope associated with dizziness. She also described double vision in her peripheral visual fields and episodes of gait disturbances causing her to have to lower herself to the ground on multiple occasions, without loss of consciousness. Her neurologic examination demonstrated mildly ataxic finger-to-nose testing and concern for peripheral field vision loss. Intake lab work revealed a blood glucose of 725 mg/dL and a hemoglobin A1C of 13.4%. Initial neuroimaging with computed tomography was unremarkable. Though her symptoms were thought to be due to severe hyperglycemia, an MRI brain was obtained due to abnormal neurologic examination. MRI demonstrated a 1.9 cm pituitary macroadenoma abutting the optic chiasm with concern for hemorrhage. Subsequent lab evaluation determined the pituitary macroadenoma non-functional with TSH, free T4, free T3, AM cortisol, AM ACTH, and prolactin all within normal limits. Her hyperglycemia was treated with insulin with clinical improvement in all regards except visual symptoms. She was deemed safe for discharge with neurosurgical follow-up regarding surgical removal of her macroadenoma. Conclusion: Although hyperglycemia may present with broad symptoms including vague neurologic symptoms, it is critical to keep a broad differential diagnosis when atypical symptoms such as persistent vision changes and gait disturbances are present, especially after improvement in glycemic control has been obtained. A low threshold should be held for obtaining neuroimaging, as it is prudent to rule-out life-threatening causes of neurologic dysfunction.
Antipsychotic Medication Use Delays Diagnosis of Acromegaly
Authors: Rosner Matthew Norman , Rao Ambika
Published: May 2021

Background: Elevated prolactin level from use of antipsychotic medication masked the diagnosis of acromegaly.

Clinical Case: A 29-year old woman presented with galactorrhea, metrorrhagia, menorrhagia, 50 lb. weight gain, hirsutism, insomnia, and breast engorgement for one year. She was evaluated by gynecology and treated with a combination estrogen/progesterone pill, and then switched to a patch. For several years she had been on haloperidol and various phenothiazine medications for paranoid schizophrenia. Initial evaluation included elevated prolactin level of 94.46 ng/ml (5.2-26.25 ng/mL). Differential diagnosis of PCOS versus medication induced hyperprolactinemia was considered. Patient was started on metformin and referred to nutrition for weight management. MRI scan ordered by her primacy care physician revealed a 13 x 12 x 10 mm pituitary mass with intrinsic mixed density and homogenous enhancement. Visual field testing revealed no deficits. On further testing: IgF-1 432 ng/ml (53-331 ng/ml), TSH 0.85 uIU/mL (0.35-4.94 IUI/ml), ACTH 22 pg/mL (6-50 pg/ml), cortisol 7.7 ug/dl (4.3-22.4 mcg/dl). Differential diagnosis of stalk compression versus functioning adenoma was considered and cabergoline was initiated after she was seen by neurosurgery and declined surgical intervention. She was noncompliant with cabergoline. Surveillance over next two years showed no increase in tumor size on MRI or change in hormone levels. She was then lost to follow up for a year and presented with increased diaphoresis and swelling of her hands. Prolactin three years after diagnosis decreased to 50.59 ng/mL and IgF-1 to 639 ng/ml. She was then referred to endocrinology. Oral glucose tolerance test did not suppress growth hormone below 1.9 ng/ml. Her cortisol suppressed to 0.1 mcg/dl with 1 mg dexamethasone. A diagnosis of Acromegaly was confirmed. She was referred for neurosurgical evaluation and scheduled for transsphenoidal hypophysectomy. She failed to arrive for scheduled surgery. Repeat MRI five years after diagnosis showed a 13 x 16 x 20 mm pituitary adenoma with IgF-1 level of 1011 ng/ml. Octreotide LAR 20 mg monthly was initiated. After two months of injections: prolactin 69.36 ng/ml, IgF-1 778 ng/ml. Repeat visual field testing showed mild constriction in the superior and inferior temporal fields. Patient was scheduled for MRI and surgical procedure but was lost to follow-up.

Conclusion: Elevation in prolactin levels in conjunction with antipsychotic medications and diagnosis of schizophrenia masked the early diagnosis of Acromegaly in this patient.
Cerebrospinal Fluid Leak After Medical Management of Prolactinoma
Authors: Pechman Amanda , Manavalan Anjali , Kishore Preeti
Published: May 2021

Introduction: Dopaminergic agonists such as cabergoline are commonly used to treat prolactinomas, and often lead to significant adenoma shrinkage. Rarely, macroprolactinomas may invade the sphenoid sinus and pose a therapeutic challenge, as treatment with dopamine agonists and subsequent tumor shrinkage can lead to a cerebrospinal fluid (CSF) leak.

Clinical Case: A 32-year-old female with no prior medical history presented to the emergency room of an outside hospital for headaches and visual changes. MRI demonstrated a 4.3 x 4.0 x 3.0 cm sellar mass with extension into the right cavernous sinus and sphenoid sinus, and prolactin was elevated to 1,859 ng/ml (3.4 - 21.4). Cabergoline was initiated at a dose of 0.25 mg twice a week with a decrease in her prolactin to 646 ng/ml one month later. Six months after initiation of therapy she transferred her care to our institution and presented for a routine endocrinology visit. She complained of rhinorrhea worse when upright that had begun two weeks prior to this encounter. Urgent MRI of the sella revealed an interval decrease in the size of the sellar mass to 2.1 x 2.7 x 1.5 cm and a fluid collection extending from the sella turcica to the left sphenoid sinus consistent with a CSF leak, and labs showed a prolactin level of 169 ng/ml. Cabergoline was discontinued and the patient was admitted to the hospital for treatment with antibiotics and acetazolamide. She underwent lumbar drain placement while awaiting definitive surgical management, which was delayed due to the COVID-19 pandemic. While off cabergoline, the sellar mass increased in size and her prolactin rose to 1,449 ng/ml, with no effect on the optic chiasm and visual fields. Three months later, she underwent transsphenoidal debulking of the pituitary mass with repair of the sellar floor and removal of lumbar drain. Her postoperative course was uncomplicated and she resumed cabergoline 0.25 mg twice weekly. A prolactin measured two weeks postoperatively was 337 ng/ml. Tumor histopathology revealed a 1.3 x 0.4 x 0.3 cm pituitary adenoma. Immunohistochemistry stained positive for chromogranin A, synaptophysin, and prolactin only. An MRI of the sella and prolactin level will be repeated at her next endocrinology visit.

Conclusions: CSF leak is a rare but serious complication from tumor shrinkage after medical management of large invasive prolactinomas, and patients with such tumors should be monitored closely for its development. CSF leaks can increase a patient’s risk for meningitis and subsequent morbidity and mortality. The management of patients with large prolactinomas with local invasion should involve a multidisciplinary approach for decision making, extensive patient education, and close follow up in order to identify and treat a CSF leak should it develop.
Clinically Functioning FSH-Secreting Pituitary Adenoma
Authors: AlNasrallah Noor , Almurshed Maryam , Aljenaee Khaled , Hajji Sulaiman
Published: May 2021

Background: Gonadotroph adenomas are the most common type of non-functioning pituitary adenomas. However functioning gonadotroph adenomas are rare with only a few cases reported in literature.

Clinical Case: A 42-year old man was admitted as a case of COVID-19 when an incidental finding of a pituitary gland mass was detected. Upon evaluation, the patient was found to have blurred vision and dizziness since few years, however no history of headache, seizures, visual field deficit were reported, nor clear symptoms related to anterior pituitary hypo- or hyperfunction. Pituitary MRI revealed a pituitary gland mass measuring 30x23x22 mm expanding the pituitary fossa and extending to suprasellar cistern. Initial laboratory investigations revealed low free T3 (1.8 pmol/L, normal: 3.1-6.8 pmol/L) and low free T4 (6.6 pmol/L, normal: 12-22 pmol/L) with normal TSH (3.54 mIU/L, normal: 0.27-4.2 mIU/L), high FSH (31.4 IU/L, normal: 1-18 IU/L), and high total testosterone (50.31 nmol/L, normal 8.3-32.9 nmol/L). Testicular ultrasound showed bilateral enlarged testicles (right testis 38.14 cm3 and left testis 33.38 cm3). Visual Field testing revealed monocular superior temporal defect affecting only the left eye. Patient was treated medically with levothyroxine and hydrocortisone and surgically by minimally invasive transsphenoidal resection of pituitary adenoma. Histopathology assessment confirmed the diagnosis of gonadotroph secreting adenoma, with positive immunostain for FSH. At 6 weeks postoperatively, insulin tolerance test was performed after which hydrocortisone was only prescribed during periods of stress. Anterior pituitary profile was repeated which revealed normalization of free T4 (14.3 pmol/L) and FSH (1.2 IU/L) and decrease in testosterone (0.66 nmol/L). Assessment of visual field revealed marked improvement of previous findings. Pituitary MRI was also repeated with findings of enhancing soft tissue remnants. At 12 weeks postoperatively, anterior pituitary profile revealed normalization of testosterone (11.3 nmol/L). Testicular ultrasound was repeated and showed a marked decrease in testicular volume (right testis 17.93 cm3 and left testis 21.13 cm3).

Conclusion: Functional gonadotroph adenomas are a rare subtype of pituitary adenomas which pose a diagnostic challenge and require meticulous clinical evaluation and multidisciplinary therapeutic approach.
Headache, Pituitary Apoplexy and Diabetes insipidus: Suspect Necrotizing Hypophysitis
Authors: Panthi Sujata , Miles John M , Graves Leland
Published: May 2021

Background: Necrotizing hypophysitis (NH) is an exceedingly rare clinical entity; we found only 5 reported cases in a literature review. CASE: A 34-year-old female with a history of primary hypothyroidism developed a headache, double vision, nausea, vomiting, decrease in appetite, galactorrhea, and weight loss. An MRI of the brain showed a 0.9 cm T2 hyperintense pituitary lesion with features suggestive of cystic degeneration or apoplexy of an adenoma There was effacement of the optic nerve anterior to the optic chiasm. Eye examination revealed optic nerve edema and a cranial nerve VI palsy. Laboratory studies revealed a normal prolactin 20.0 (3.3-26.7 ng/ml), and IGF-1 189 (RR: 66 - 303 ng/ml). The pituitary-adrenal axis and pituitary-gonadal axis appeared intact. After short treatment with dexamethasone, her primary symptom of severe headache improved but she later developed polyuria. The evaluation confirmed diabetes insipidus. Her headache worsened after the completion of the brief course of steroids. After a repeat MRI showed mild interval enlargement of the hypo-enhancing posterior pituitary, she underwent transsphenoidal pituitary tumor surgery. A biopsy showed necrotizing inflammation with foci of acute necrosis accompanied by polymorphonuclear cells. There was no granuloma seen. Special stains were negative for fungal, acid-fast, and bacterial organisms. Laboratory studies revealed negative ANA, negative ANCA, PR-3 Ab, MPO Ab, and anti-TPO Abs. The ACE enzyme was elevated at 85 (RR: 8-53 U/l), but the repeat test was within the normal range. A chest CT was not suggestive of pulmonary sarcoidosis. A post-surgery MRI demonstrated a decrease in pituitary size. Her headache worsened with a reduction in prednisone below 20 mg daily. In order to attempt further reduction in prednisone, azathioprine was started as a steroid-sparing agent. Titration of azathioprine up to 175 mg allowed prednisone to be reduced to the replacement dose of prednisone 5 mg daily. An MRI showed the pituitary mass to be unchanged. DISCUSSION: Hypophysitis is associated with diffuse enlargement and dysfunction of the pituitary gland. This case of NH is of unclear cause. There was no evidence of other inflammatory causes such as sarcoidosis, IgG4-related disease, ANCA-associated vasculitis, or infectious cause. As with typical lymphocytic hypophysitis, NH can present with sudden-onset hypopituitarism, neural compression, diabetes insipidus, and radiologic features of ischemic pituitary apoplexy. Signs and symptoms at diagnosis, as well as pituitary hormone abnormalities, vary on the degree of pituitary involvement. Pituitary adenoma commonly does not cause diabetes Insipidus. Radiologic findings consistent with ischemic pituitary apoplexy coupled with the presence of diabetes insipidus is suggestive of NH. A definitive diagnosis of NH, however, requires histopathology.