Glucocorticoid Deficiency Research Articles

Mitochondrial Nicotinamide Nucleotide Transhydrogenase: Role in Energy Metabolism, Redox Homeostasis, and Cancer.

Significance: Dimeric nicotinamide nucleotide transhydrogenase (NNT) is embedded in the mitochondrial inner membrane and couples the conversion of NADP+/NADH into NADPH/NAD+ to mitochondrial matrix proton influx. NNT was implied in various cancers, but its physiological role and regulation still remain incompletely understood. Recent Advances: NNT function was analyzed by studying: (1) NNT gene mutations in human (adrenal) glucocorticoid deficiency 4 (GCCD4), (2) Nnt gene mutation in C57BL/6J mice, and (3) the effect of NNT knockdown/overexpression in (cancer) cells. In these three models, altered NNT function induced both common and differential aberrations. Critical Issues: Information on NNT protein expression in GCCD4 patients is still scarce. Moreover, NNT expression levels are tissue-specific in humans and mice and the functional consequences of NNT deficiency strongly depend on experimental conditions. In addition, data from intact cells and isolated mitochondria are often unsuited for direct comparison. This prevents a proper understanding of NNT-linked (patho)physiology in GCCD4 patients, C57BL/6J mice, and cancer (cell) models, which complicates translational comparison. Future Directions: Development of mice with conditional NNT deletion, cell-reprogramming-based adrenal (organoid) models harboring specific NNT mutations, and/or NNT-specific chemical inhibitors/activators would be useful. Moreover, live-cell analysis of NNT substrate levels and mitochondrial/cellular functioning with fluorescent reporter molecules might provide novel insights into the conditions under which NNT is active and how this activity links to other metabolic and signaling pathways. This would also allow a better dissection of local signaling and/or compartment-specific (i.e., mitochondrial matrix, cytosol, nucleus) effects of NNT (dys)function in a cellular context. Antioxid. Redox Signal. 41, 927-956.

12203 FDXR Variants Cause Adrenal Insufficiency And Atypical Sexual Development

Abstract Disclosure: E. Pignatti: None. J. Slone: None. M. Gomez-Cano: None. T.M. Campbell: None. J. Vu: None. K. Sauter: None. A.V. Pandey: None. M. Alonso-Riaño: None. F. Martínez-Azorín: None. D. Neilson: None. N. Longo: None. T. Du Toit: None. C.D. Voegel: None. T. Huang: None. C.E. Flueck: Consulting Fee; Self; Novo Nordisk. Grant Recipient; Self; Novo Nordisk, Sandoz, Pfizer, Inc., Merck. Speaker; Self; Sandoz. Background. Biallelic variants in the mitochondrial flavoprotein ferredoxin—NADP(+)-reductase (FDXR) have been associated with a progressive neuropathic mitochondriopathy named FDXR-Related Mitochondriopathy (FRM). FRM patients often experience worsening or demise following stress, but the cause is unclear. Ambiguous genitalia at birth have now been observed in some FRM patients. As FDXR is an essential partner for several steroid enzymes, we explored the impact of FDXR variants on steroid biosynthesis. Methods. To investigate whether the abnormal stress response of many FRM patients is caused by glucocorticoid deficiency, and whether observed ambiguous genitalia is related to androgen excess, we investigated the impact of FDXR variants on adrenal steroidogenesis. Clinical, genetic, biochemical, and functional studies were performed in index patients, steroidogenic cell lines reprogrammed from patient fibroblasts, and a novel disease mouse model. Results. Two paradigmatic female FRM patients carrying novel homozygous FDXR mutation p.G437R with ambiguous genitalia at birth and sudden death in the first year of life had cortisol deficiency and androgen excess compatible with 11-hydroxylase deficiency. Steroidogenic FDXR-variant cells lines reprogrammed from three patients’ fibroblasts also displayed deficient mineralocorticoid and glucocorticoid production. Likewise, Fdxr-variant mice allelic to the severe p.R386W human variant, showed reduced progesterone and corticosterone production. Conclusions. Our comprehensive studies show that FDXR variants in FRM patients may cause compensated, but possibly life-threatening adrenocortical insufficiency in stress by affecting adrenal glucocorticoid and mineralocorticoid synthesis through direct enzyme inhibition, most likely in combination with disturbed mitochondrial redox balance. Presentation: 6/1/2024

Adrenocortical insufficiency after bilateral adrenal hemorrhage due to anticoagulation and chronic immunothrombocytopenia.

Adrenocortical insufficiency is defined as the clinical manifestation of chronic glucocorticoid and/or mineralocorticoid deficiency due to failure of the adrenal cortex. It may result in an adrenal crisis, which is a life-threatening disease; thus, prompt initiation of therapy with hydrocortisone is necessary. Symptoms such as hypotension, weight loss, or fatigue are not specific, which is why diagnosis is delayed in many cases. Our patient suffered from immune thrombocytopenia (ITP), an acquired thrombocytopenia caused by an autoimmune reaction against platelets and megakaryocytes. Primary ITP, in which no triggering cause can be identified, must be distinguished from secondary forms (e.g. in the context of systemic autoimmune diseases, lymphomas, or (rarely) by drugs). Patients may be asymptomatic at presentation or may present with a range of mild mucocutaneous to life-threatening bleeding. Here, we report on a 43-year-old woman who had developed adrenocortical insufficiency due to bilateral hemorrhage in the adrenal glands. Because of anticoagulation with phenprocoumon after pulmonary embolism and thrombocytopenia on the basis of ITP, the patient had an increased risk of bleeding. Due to the nonspecific and ambiguous symptoms of adrenocortical insufficiency, prompt diagnosis remains a challenge. Hypocortisolism or adrenal crisis with nonspecific symptoms, especially abdominal and gastrointestinal, is often misinterpreted. Diagnosis of adrenal insufficiency is often delayed because of the initial ambiguous presentation; physicians must be aware to avoid adrenal crisis. Especially in patients with several risk factors for bleeding, unusual bleeding manifestations, such as adrenal hemorrhage, must be considered. Immediate treatment is necessary by substituting hydrocortisone in a higher dosage, and in most cases, fludrocortisone. During the course of treatment, the amount of hydrocortisone can be reduced to a substitution dosage (15-25 mg/day divided into two to three doses/day). Fludrocortisone should be continued at a dosage of 0.05-0.1 mg/day, depending on blood pressure and sodium and potassium levels. All patients should carry a medical alert notification or a steroid emergency card. In the case of trauma, surgery, or other stressful events, hydrocortisone must be administered in higher dosages (e.g. 100 mg i.v.).

Comparison between typical primary and eunatraemic, eukalaemic hypoadrenocorticism: 92 cases

BackgroundNaturally occurring hypoadrenocorticism is an uncommon endocrine disorder in dogs but has significant morbidity and mortality. Some dogs present with apparent glucocorticoid deficiency alone as evidenced by eunatraemia and eukalaemia. Few studies have compared dogs with hypoadrenocorticism with or without electrolyte disturbances and there are no large case series of affected dogs from Ireland.MethodsRetrospective observational study.ResultsNinety-two cases diagnosed with hypoadrenocorticism subdivided into those with supportive electrolyte disturbances (Group 1; n = 72) and those without (Group 2; n = 20). Dogs in Group 1 were significantly (p = 0.001) younger (4.0 (3.0–6.0) years) than dogs in Group 2 (6.0 (4.75–8.25) years). Dogs in Group 1 presented significantly more commonly with vomiting (Group 1: 52/71 (73.2%), Group 2: 6/20 (30.0%); p < 0.001), total hyperproteinaemia (Group 1: 21/71 (29.6%), Group 2: 1/20 (5.0%); p = 0.023), increased urea (Group 1: 52/72 (72.2%), Group 2: 5/20 (25.0%); p < 0.001), increased creatinine (Group 1: 31/72 (43.1%), Group 2: 3/20 (15.0%); p = 0.021) and hyperphosphataemia (Group 1: 40/71 (56.3%), Group 2: 2/20 (10.0%); p < 0.001), and significantly less commonly with reticulocytosis (Group 1: 4/38 (10.5%), Group 2: 5/13 (38.5%), p = 0.023). An undetectable basal aldosterone concentration had a positive predictive value of 94.3% for diagnosing undetectable post-ACTH aldosterone concentration. Of the thirteen dogs in Group 2 that had aldosterone concentrations measured and secondary disease excluded, 7 (53.8%) had or subsequently developed evidence of aldosterone deficiency, although not always with electrolyte abnormalities.ConclusionsDogs with hypoadrenocorticism from Ireland are similar to other reported cases. An undetectable basal aldosterone concentration is highly predictive of mineralocorticoid deficiency. Dogs with apparent glucocorticoid deficiency alone can progress to more typical disease and should be monitored appropriately.

Corticotropin-releasing hormone deficiency results in impaired analgesic response during CFA-induced inflammation

PurposeCorticotropin-releasing hormone (CRH) plays an important role in relief of pain by releasing analgesia-associated molecules in several inflammatory states. During inflammation, peripheral CRH acts on cells of the immune system to stimulate the local expression of proopiomelanocortin (POMC) and the production of β-endorphin, which in turn binds to opioid receptors on sensory neurons to produce antinociception. In the present study, we further investigated the role of endogenous CRH in inflammatory pain by determining the effects of Crh-deficiency on this process.MethodsFor this purpose, we used Crh-deficient (Crh-/-) mice and their wildtype (Crh + / +) littermates in the CFA (Complete Freund’s Adjuvant)-induced inflammatory pain model. Pain thresholds were evaluated with the Hargreaves apparatus.ResultsOur experiments showed that Crh deficiency led to increased pain response, which was associated with decreased POMC mRNA levels in locally inflamed paws of these mice. Furthermore, Crh-/- mice had higher paw edema than Crh + / + mice. Histological evaluation of inflamed paw tissues revealed increased inflammatory response in Crh-/- mice. Protein levels of proinflammatory cytokines, such as IL-6, TNF-α, and IL-1β, were higher in inflamed tissue of Crh-/- mice compared to wildtype mice. Corticosterone replacement increased the pain threshold of Crh-/- mice, restored their paw volume to the levels of wildtype mice, and significantly reduced their proinflammatory cytokine levels. Furthermore, glucocorticoid administration significantly increased POMC mRNA expression in the inflamed paw.ConclusionOur data suggest that genetic deficiency of CRH is associated with increased pain. This effect is likely attributable to the accompanying glucocorticoid insufficiency and is in part mediated by opioids expressed locally.

Impact of Glucocorticoid Replacement Therapy on Nocturnal Hypoglycemia in Adrenal Insufficiency: An Analysis of Multiple Case Studies.

Adrenal insufficiency (AI) is a hormonal disorder characterized by insufficient glucocorticoid production. Nocturnal hypoglycemia (NH) occurs in patients with AI. However,the effect of glucocorticoid replacement therapy (GCRT) on AI and NH remains unclear. This study aimed to investigate the relationship between AI and NH by evaluating the impact of GCRT on NH in patients newly diagnosed with AI. The present study was conducted between October 2018 and December 2022 at the Department of Diabetes, Metabolism and Endocrinology of the Tokyo Rosai Hospital, Japan. In total, 15 patients aged ≥18 years with newly diagnosed AI or NH were included in this study. The NH frequency was measured using continuous glucose monitoring (CGM). The primary outcome was the change in NH frequency before and after the GCRT intervention. GCRT significantly decreased NH frequency. Severe NH frequency and minimum nocturnal glucose levels changed significantly while fasting blood glucose and glycated hemoglobin levels did not change significantly. GCRT intervention improved CGM profiles' time below range, time in range, and average daily risk range. The present study suggests that GCRT can help newly diagnosed patients with AI manage NH. These findings show that CGM can detect NH in patients with newly diagnosed AI, determine the optimal GCRT dosage, and hence prevent an impaired quality of life and even serious adverse effects in these patients. Further large multicenter studies should validate these findings and delve deeper into the mechanistic link between AI and NH.

Genetic Disruption of cyp21a2 Leads to Systemic Glucocorticoid Deficiency and Tissues Hyperplasia in the Teleost Fish Medaka (Oryzias latipes).

cytochrome P-450, 21-hydroxylase (cyp21a2), encodes an enzyme required for cortisol biosynthesis, and its mutations are the major genetic cause of congenital adrenal hyperplasia (CAH) in humans. Here, we have generated a null allele for the medaka cyp21a2 with a nine base-pair insertion which led to a truncated protein. We have observed a delay in hatching and a low survival rate in homozygous mutants. The interrenal gland (adrenal counterpart in teleosts) exhibits hyperplasia and the number of pomca-expressing cells in the pituitary increases in the homozygous mutant. A mass spectrometry-based analysis of whole larvae confirmed a lack of cortisol biosynthesis, while its corresponding precursors were significantly increased, indicating a systemic glucocorticoid deficiency in our mutant model. Furthermore, these phenotypes at the larval stage are rescued by cortisol. In addition, females showed complete sterility with accumulated follicles in the ovary while male homozygous mutants were fully fertile in the adult mutants. These results demonstrate that the mutant medaka recapitulates several aspects of cyp21a2-deficiency observed in humans, making it a valuable model for studying steroidogenesis in CAH.

Glucocorticoid-induced adrenal insufficiency and glucocorticoid withdrawal syndrome: Two sides of the same coin.

Diseases of the adrenal glands can lead to primary adrenal insufficiency, and suppression of the hypothalamic-pituitary-adrenal axis can cause secondary adrenal insufficiency (adrenal suppression). The most common cause of adrenal suppression is exogenous steroids, a condition recently termed glucocorticoid-induced adrenal insufficiency (GIAI). Similarly, weaning from high doses of glucocorticoids or giving insufficient glucocorticoid replacement after curative surgery for endogenous hypercortisolism (Cushing syndrome) can lead to glucocorticoid withdrawal syndrome, which overlaps with GIAI.

Stress-induced urgent conditions in endocrinology

The literature review deals with the influence of chronic stress on the functioning of the endocrine system. The most significant pathological conditions induced by chronic stress are decompensation of carbohydrate metabolism, disorders of the thyroid gland, reproductive system, depressive disorders, electrolyte disorders. The significance of these pathologies is determined by a pronounced decrease in the quality of life, the occurrence of severe complications, and an increase in mortality. The development of hyperglycemia during the acute period of the disease is an established connection between the stress system and glucose homeostasis, which is confirmed by the development of stress hyperglycemia and increased morbidity. Glucocorticoids have a contrainsular effect, and the significance of their impact on carbohydrate metabolism is due to the pleiotropic action: induction of insulin resistance, stimulation of gluconeogenesis, increase in the blood content of glucagon due to dysregulation of its synthesis and secretion. The effects of stress on the thyroid gland are determined by its duration, characterized by the activation of the pituitary-thyroid system in the acute period and its suppression during long-term and chronic stress. The contribution of the stress component and glucocorticoid deficiency to the development of autoimmune thyroid diseases due to the suppression of cellular and strengthening of humoral immunity is discussed. Under conditions of psychological stress, the development of hypothyroidism was noted in emigrants with anxiety-depressive syndrome and insomnia, as evidenced by a decrease in the concentration of thyroid hormones. A decreased thyroid status in these patients is associated with the severity of chronic stress. Research data show that chronic stress can significantly change a person’s thyroid status, leading to the development of triiodothyronine deficiency syndrome and an increase in the production of inactive reverse triiodothyronine, inducing the development of hypothyroidism. It is possible that the pronounced growth of subclinical forms of hypothyroidism, noted in many countries of the world, is a consequence of chronic stress. The hypothalamic-pituitary-adrenal axis is involved in the formation of stress-induced reactions almost immediately after exposure to the stressor. Stress-induced changes in the endocrine system arise and develop in close connection, forming a vicious circle of pathological changes and emergencies.

Establishment of human induced pluripotent stem cell line SDQLCHi029-A from one Type 1 familial glucocorticoid deficiency patient carrying compound heterozygote mutations in MC2R gene

Type 1 familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder due to variation of the melanocortin-2-receptor (MC2R) gene. Induced pluripotent stem cell (iPSC) line SDQLCHi029-A was successfully generated from peripheral blood mononuclear cells obtained from a 5-day-old girl with MC2R mutations (c.428C > T and c.409C > T). The iPSC line showed genetically stable and matched the donor’s PBMCs. displayed a normal karyotype, expressed high pluripotent markers, and exhibited differentiation potential of three germ layers in vitro. The iPSC line could be a good model to study the pathogenesis of FGD type 1 and screen new drugs for the disease.

Plasma steroid concentrations reflect acute disease severity and normalise during recovery in people hospitalised with COVID-19.

Endocrine systems are disrupted in acute illness, and symptoms reported following coronavirus disease 2019 (COVID-19) are similar to those found with clinical hormone deficiencies. We hypothesised that people with severe acute COVID-19 and with post-COVID symptoms have glucocorticoid and sex hormone deficiencies. Samples were obtained for analysis from two UK multicentre cohorts during hospitalisation with COVID-19 (International Severe Acute Respiratory Infection Consortium/World Health Organisation [WHO] Clinical Characterization Protocol for Severe Emerging Infections in the UK study), and at follow-up 5 months after hospitalisation (Post-hospitalisation COVID-19 study). Plasma steroids were quantified by liquid chromatography-mass spectrometry. Steroid concentrations were compared against disease severity (WHO ordinal scale) and validated symptom scores. Data are presented as geometric mean (SD). In the acute cohort (n = 239, 66.5% male), plasma cortisol concentration increased with disease severity (cortisol 753.3 [1.6] vs. 429.2 [1.7] nmol/L in fatal vs. least severe, p < .001). In males, testosterone concentrations decreased with severity (testosterone 1.2 [2.2] vs. 6.9 [1.9] nmol/L in fatal vs. least severe, p < .001). In the follow-up cohort (n = 198, 62.1% male, 68.9% ongoing symptoms, 165 [121-192] days postdischarge), plasma cortisol concentrations (275.6 [1.5] nmol/L) did not differ with in-hospital severity, perception of recovery, or patient-reported symptoms. Male testosterone concentrations (12.6 [1.5] nmol/L) were not related to in-hospital severity, perception of recovery or symptom scores. Circulating glucocorticoids in patients hospitalised with COVID-19 reflect acute illness, with a marked rise in cortisol and fall in male testosterone. These findings are not observed 5 months from discharge. The lack of association between hormone concentrations and common post-COVID symptoms suggests steroid insufficiency does not play a causal role in this condition.

Expanding the Phenotype of Congenital Glucocorticoid Deficiency: An Iranian Patient with Cholestasis due to Pathogenic Variants in the MC2R Gene.

Familial glucocorticoid deficiency is caused by variants in the MC2R and MRAP genes. We report an Iranian patient with congenital glucocorticoid deficiency and cholestasis due to pathogenic variants in the MC2R gene. This is the first documented case of a patient with conditions. Clinical evaluations and lab assessments were conducted on a six-month-old male infant. Next-generation sequencing identified the genetic causes of the disease, and Sanger sequencing confirmed the variants through segregation analysis. The clinical presentation included prolonged jaundice, progressive skin hyperpigmentation, seizures, fever, and a large umbilical hernia. Two variants in the MC2R gene, c.560delT and c.676G > C, were detected and classified as pathogenic and likely pathogenic, respectively. The cooccurrence of cholestasis and glucocorticoid deficiency illustrates the clinical heterogeneity caused by MC2R variants. The prevalence of c.560delT and c.676G > C between Iranian populations suggests these variants may be common. The high frequency of c.560delT could be attributed to a founder effect.

Familial Glucocorticoid Deficiency: the changing landscape of an eponymous syndrome.

Familial Glucocorticoid Deficiency encompasses a broad spectrum of monogenic recessive disorders that theoretically solely abrogate cortisol biosynthesis. In reality, delineating clear genotype-phenotype correlations in this disorder is made complicated by marked phenotypic heterogeneity even within kindreds harbouring identical variants. Phenotypes range from isolated glucocorticoid insufficiency to cortisol deficiency plus a variety of superimposed features including salt-wasting and hypoaldosteronism, primary hypothyroidism, hypogonadism and growth defects. Furthermore, mutation type, domain topology and perceived enzyme activity do not always predict disease severity. Given the high burden of disease and implications of a positive diagnosis, genetic testing is crucial in the management of patients warranting detailed delineation of genomic variants including viable functional studies.

Clinical characteristics and genetic analysis of two children with Familial glucocorticoid deficiency type 1 due to variants of MC2R gene

To improve the recognition of Familial glucocorticoid deficiency type 1 (FGD1) due to variants of melanocortin 2 receptor (MC2R) gene. Two children with FGD1 diagnosed at the Henan Children's Hospital respectively in 2019 and 2021 were selected as the study subjects. Clinical data, treatment, follow-up and results of genetic testing were collected and retrospectively analyzed. Whole exome sequencing revealed that both children had harbored compound heterozygous variants of the MC2R gene, including c.433C>T (p.R145C) and c.710T>C (p.L237P) in child 1, and c.145delG (p.V49Cfs*35) and c.307G>A (p.D103N) in child 2, among which c.710T>C (p.L237P) and c.145delG (p.V49Cfs*35) were unreported previously. FGD1 is clinically rare, and genetic sequencing is crucial for the definite diagnosis. Discovery of the and novel variants has enriched the mutational spectrum of the FGD1 gene.

Major immunophenotypic abnormalities in patients with primary adrenal insufficiency of different etiology.

Patients with primary adrenal insufficiency (PAI) suffer from increased risk of infection, adrenal crises and have a higher mortality rate. Such dismal outcomes have been inferred to immune cell dysregulation because of unphysiological cortisol replacement. As the immune landscape of patients with different types of PAI has not been systematically explored, we set out to immunophenotype PAI patients with different causes of glucocorticoid (GC) deficiency. This cross-sectional single center study includes 28 patients with congenital adrenal hyperplasia (CAH), 27 after bilateral adrenalectomy due to Cushing's syndrome (BADx), 21 with Addison's disease (AD) and 52 healthy controls. All patients with PAI were on a stable GC replacement regimen with a median dose of 25 mg hydrocortisone per day. Peripheral blood mononuclear cells were isolated from heparinized blood samples. Immune cell subsets were analyzed using multicolor flow cytometry after four-hour stimulation with phorbol myristate acetate and ionomycin. Natural killer (NK-) cell cytotoxicity and clock gene expression were investigated. The percentage of T helper cell subsets was downregulated in AD patients (Th1 p = 0.0024, Th2 p = 0.0157, Th17 p < 0.0001) compared to controls. Cytotoxic T cell subsets were reduced in AD (Tc1 p = 0.0075, Tc2 p = 0.0154) and CAH patients (Tc1 p = 0.0055, Tc2 p = 0.0012) compared to controls. NKCC was reduced in all subsets of PAI patients, with smallest changes in CAH. Degranulation marker CD107a expression was upregulated in BADx and AD, not in CAH patients compared to controls (BADx p < 0.0001; AD p = 0.0002). In contrast to NK cell activating receptors, NK cell inhibiting receptor CD94 was upregulated in BADx and AD, but not in CAH patients (p < 0.0001). Although modulation in clock gene expression could be confirmed in our patient subgroups, major interindividual-intergroup dissimilarities were not detected. In patients with different etiologies of PAI, distinct differences in T and NK cell-phenotypes became apparent despite the use of same GC preparation and dose. Our results highlight unsuspected differences in immune cell composition and function in PAI patients of different causes and suggest disease-specific alterations that might necessitate disease-specific treatment.

Primary adrenal insufficiency with muco-candidiasis- a rare cause of familial glucocorticoid deficiency due to thioredoxin reductase deficiency

Primary adrenal insufficiency with muco-candidiasis- a rare cause of familial glucocorticoid deficiency due to thioredoxin reductase deficiency

Congenital dysfunction of the adrenal cortex on the background of 11β-hydroxylase deficiency

Congenital adrenal dysfunction (CHD) is a variant of hereditary fermentopathies that lead to a violation of the synthesis of cortisol in the adrenal cortex. Late diagnosis of this pathology significantly impairs the quality of life of patients, and in some cases can lead to fatal consequences. The most common form of CHD, occurring in more than 90% of cases, is due to a deficiency of 21-hydroxylase, which is responsible for the synthesis of deoxycorticosterone and 11-deoxycortisol. The prevalence of this form is 1:10,000 to 1:20,000 newborns in the world, in Russia — 1:9500. In second place is the deficiency of 11β-hydroxylase (hypertonic form of HCHD), which affects the synthesis of cortisol. This form of the disease occurs in about 1:100,000 newborns in the world, and in Russia the prevalence of this form is unknown [1].The cause of any form of CHD is mutations in the genes responsible for the synthesis of enzymes or transport proteins involved in the synthesis of cortisol. 11β-hydroxylase is synthesized in the zona fasciculata of the adrenal cortex and is regulated by adrenocorticotropic hormone by a negative feedback mechanism. 11β-hydroxylase deficiency (hypertonic form of HCHD) develops as a result of mutations in the CYP11B gene located on chromosome 8. This mutation blocks the enzymatic conversion of 11-deoxycortisol to cortisol, as well as deoxycorticosterone (DOC) to corticosterone, leading to the accumulation of DOC, which has mineralocorticoid activity. It is the excess of deoxycorticosterone that causes an increase.In 21-hydroxylase deficiency, the clinical picture is primary adrenal insufficiency (deficiency of glucoand mineralocorticoids). The clinical picture of mineralocorticoid deficiency includes salt loss syndrome: arterial hypotension, tachycardia, adynamia, fibrillary muscle twitching, dryness and decreased skin turgor, cyanosis and marbling of the skin. Glucocorticoid deficiency is manifested by muscle weakness, fatigue, hyperpigmentation of the skin. With a deficiency of 11β-hydroxylase, due to an increase in the level of deoxycorticosterone, there is no deficiency of mineralocorticoid activity, on the contrary, against the background of clinical signs of glucocorticoid insufficiency, an increase in blood pressure may be observed. Hyperandrogenism is characteristic of both forms of CHD. Symptoms of hyperandrogenism in females: virilization of the external genitalia, amenorrhea, severe alopecia and hirsutism. In undiagnosed cases of CHD, patients with a female karyotype have a male phenotype.

Single-Exon Deletions of ZNRF3 Exon 2 Cause Congenital Adrenal Hypoplasia.

Primary adrenal insufficiency (PAI) is a life-threatening condition characterized by the inability of the adrenal cortex to produce sufficient steroid hormones. E3 ubiquitin protein ligase zinc and ring finger 3 (ZNRF3) is a negative regulator of Wnt/β-catenin signaling. R-spondin 1 (RSPO1) enhances Wnt/β-catenin signaling via binding and removal of ZNRF3 from the cell surface. This work aimed to explore a novel genetic form of PAI. We analyzed 9 patients with childhood-onset PAI of biochemically and genetically unknown etiology using array comparative genomic hybridization. To examine the functionality of the identified single-exon deletions of ZNRF3 exon 2, we performed three-dimensional (3D) structure modeling and in vitro functional studies. We identified various-sized single-exon deletions encompassing ZNRF3 exon 2 in 3 patients who showed neonatal-onset adrenal hypoplasia with glucocorticoid and mineralocorticoid deficiencies. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the 3 distinct single-exon deletions were commonly transcribed into a 126-nucleotide deleted mRNA and translated into 42-amino acid deleted protein (ΔEx2-ZNRF3). Based on 3D structure modeling, we predicted that interaction between ZNRF3 and RSPO1 would be disturbed in ΔEx2-ZNRF3, suggesting loss of RSPO1-dependent activation of Wnt/β-catenin signaling. Cell-based functional assays with the TCF-LEF reporter showed that RSPO1-dependent activation of Wnt/β-catenin signaling was attenuated in cells expressing ΔEx2-ZNRF3 as compared with those expressing wild-type ZNRF3. We provided genetic evidence linking deletions encompassing ZNRF3 exon 2 and congenital adrenal hypoplasia, which might be related to constitutive inactivation of Wnt/β-catenin signaling by ΔEx2-ZNRF3.

SAT297 Major Immunophenotypic Abnormalities In Patients With Primary Adrenal Insufficiency Of Different Etiology

Abstract Disclosure: H.F. Nowotny: None. T. Marchant Seiter: None. J. Ju: None. A. Gottschlich: None. H. Schneider: None. S. Zopp: None. F. Vogel: None. L. Tschaidse: None. M.K. Auer: None. C. Lottspeich: None. S. Kobold: None. S. Rothenfusser: None. F. Beuschlein: None. M. Reincke: None. L. Braun: None. N. Reisch: None. Background: Increased risk of infection, adrenal crises and a higher mortality rate have been reported for patients with primary adrenal insufficiency (PAI). Such dismal outcomes have been inferred to immune cell dysregulation as a consequence of unphysiological cortisol replacement. As the immune landscape of patients with different types of PAI has not been systematically explored, we set out to immunophenotype PAI patients with different causes of glucocorticoid deficiency. Methods: The present cross-sectional single center study includes 28 patients with congenital adrenal hyperplasia (CAH), 27 patients after bilateral adrenalectomy due to Cushing’s syndrome (BADx), 21 patients with Addison’s disease (AD) and 52 healthy controls. All patients with PAI were on a stable glucocorticoid replacement regimen using a median dose of 25 mg hydrocortisone (IQR 5 mg for BADx and AD and IQR 10 mg for CAH) per day. Heparinized blood samples were processed to isolate peripheral blood mononuclear cells. All samples were processed for analysis of immune cell subsets using multicolor flow cytometry after four-hour stimulation with phorbol myristate acetate (PMA)/Ionomycin, as well as analysis of natural killer (NK-) cell cytotoxicity and clock gene expression. Results: Analysis of immune cell profiles revealed reduced percentages of IFNγ-secreting T helper (Th1) in AD (p = 0.0024) and cytotoxic (Tc1) cells in CAH (p = 0.0055) and AD patients (p = 0.0075) compared to controls. Moreover, we observed a downregulation of the percentage of IL-4+ Th2 cells in AD patients (p = 0.0157) and Tc2 cells in AD (p = 0.0154) and CAH patients (p = 0.0012) compared to controls. Th17 and Tc17 cells were also downregulated in patients with AD compared to the other patient groups and controls (p &amp;lt; 0.0001). NK-cell cytotoxicity (NKCC) using bioluminescence measurements was reduced in all subsets of PAI patients compared to controls with the lowest change in patients with CAH (mean specific lysis 57.5% in controls, 21.7% in CAH, -0.5% in AD and -28.7% in BADx). The percentage of activated NK-cells was upregulated in BADx (p = 0.0008) and AD patients (p = 0.0348) compared to controls, also expressed as an increase in CD107a expression as a marker of NK-cell degranulation (BADx p &amp;lt; 0.0001; AD p = 0.0002). While the percentage of NK-cell activating receptor expressing NK-cells did not differ from controls, expression percentage of NK-cell inhibiting receptor CD94 was upregulated in both BADx and AD patients (p &amp;lt; 0.0001). Conclusion: In patients with three different etiologies of PAI, distinct differences in T- and NK-cell-phenotypes became apparent despite the use of same GC preparation and dose. Our results highlight so far unsuspected differences in immune cell composition and NK-cell function in PAI patients of different causes and suggest disease-specific alterations that might necessitate disease-specific treatment. Presentation: Saturday, June 17, 2023

SAT626 The Use Of Salivary Cortisol Measurement By Mass Spectrometry To Identify Low Serum Cortisol Values In Healthy Volunteers Receiving CRN04894, A Melanocortin 2 Receptor (MC2R) Antagonist

Abstract Disclosure: A. Ayala: Employee; Self; Crinetics Pharmaceuticals Inc. C. LaCerte: Employee; Self; Crinetics Pharmaceuticals Inc. R. Luo: Employee; Self; Crinetics Pharmaceuticals Inc. C. Davidson: Employee; Self; Crinetics Pharmaceuticals Inc. J. Wang: Employee; Self; Crinetics Pharmaceuticals Inc. P. Trainer: Employee; Self; Crinetics Pharmaceuticals Inc. A. Krasner: Employee; Self; Crinetics Pharmaceuticals Inc. Clinical trials of cortisol-lowering drug candidates would benefit from user-friendly methods to measure cortisol in an outpatient setting. CRN04894 is a potent, orally bioavailable, MC2R antagonist in development for the treatment of ACTH-dependent Cushing’s syndrome and congenital adrenal hyperplasia. We have previously reported results from a randomized, double-blinded, placebo-controlled (6 active:3 placebo/cohort), multiple (10-day) ascending dose (40 to 80 mg/day) study in healthy volunteers. Declines in basal and ACTH-stimulated serum cortisol levels and a median reduction of 75% in 24-hr urinary-free cortisol (UFC) were seen in the 80 mg cohort. Asymptomatic glucocorticoid deficiency (defined as 08:00 serum cortisol of &amp;lt;5 µg/dL) was the most commonly observed adverse event. Here we report the comparison of salivary cortisol measurements with serum cortisol day curves and 24-hr UFC observed in this study. We evaluated cortisol and ACTH data from the three cohorts dosed once daily (QD) at 22:00 with either 40, 60, or 80 mg. Cortisol (salivary &amp; serum) and ACTH day curves (results reported as the mean of 4 or 5 available samples collected at 22:00, 08:00, 12:00, 16:00 &amp; 20:00) studies were undertaken on days -1, 1, 4, and 9. Mean serum cortisol was moderately correlated with 24-hr UFC:creatinine ratio (ug/g) (r = 0.495) and strongly correlated (r = 0.734) with mean salivary cortisol. There was across all three doses of CRN04894 a consistent magnitude ranking change in measured cortisol analytes: serum (free plus bound) &amp;lt; saliva &amp;lt; urine, with substantial changes being seen within 24 hours of initiation of CRN04894 and no loss of cortisol suppression over 9 days despite median ACTH changes of &amp;gt;1000% from baseline (60 &amp; 80 mg doses). Time-matched individual saliva and serum cortisol values were used (without timepoint or visit restrictions) to explore the relationship between low serum cortisol and salivary levels. A saliva cutoff for cortisol deficiency was optimized for diagnostic accuracy (92.2%) using a serum cortisol value of &amp;lt;5 µg/dL as the reference. Four subjects had 08:00 serum cortisol &amp;lt;5 µg/dL and saliva cortisol data. All four cases were correctly identified as cortisol-deficient using a simultaneous saliva cortisol cutoff of &amp;lt;0.0491 µg/dL. In conclusion, CRN04894 effectively lowered cortisol measured in serum, saliva, and urine in healthy volunteers. Salivary cortisol measured by tandem mass spectrometry correlated well with serum cortisol day curves and may be useful in the diagnosis of biochemical cortisol deficiency in outpatient studies. Future studies will explore the use of salivary glucocorticoid measurement in patients with Cushing’s disease receiving CRN04894. Presentation: Saturday, June 17, 2023