Dominant Mutations in the Autoimmune Regulator AIRE Are Associated with Common Organ-Specific Autoimmune Diseases.

The autoimmune regulator (AIRE) gene is crucial for establishing central immunological tolerance and preventing autoimmunity. Mutations in AIRE cause a rare autosomal-recessive disease, autoimmune polyendocrine syndrome type 1 (APS-1), distinguished by multi-organ autoimmunity. We have identified multiple cases and families with mono-allelic mutations in the first plant homeodomain (PHD1) zinc finger of AIRE that followed dominant inheritance, typically characterized by later onset, milder phenotypes, and reduced penetrance compared to classical APS-1. These missense PHD1 mutations suppressed gene expression driven by wild-type AIRE in a dominant-negative manner, unlike CARD or truncated AIRE mutants that lacked such dominant capacity. Exome array analysis revealed that the PHD1 dominant mutants were found with relatively high frequency (>0.0008) in mixed populations. Our results provide insight into the molecular action of AIRE and demonstrate that disease-causing mutations in the AIRE locus are more common than previously appreciated and cause more variable autoimmune phenotypes.

Pulsatile Subcutaneous Hydrocortisone Replacement in Primary Adrenal Insufficiency.

Pulsatile endogenous cortisol secretion is critical for physiological glucocorticoid gene signaling. Conventional glucocorticoid replacement therapy does not mimic endogenous cortisol pulsing in primary adrenal insufficiency. In an open-labeled, two-week, nonrandomized cross-over study of five patients with adrenal insufficiency (Addison's disease in two, bilateral adrenalectomy in one, and congenital adrenal hyperplasia in two patients) we compared pulsatile and continuous cortisol pump treatment and conventional oral glucocorticoid therapy with respect to 24-h serum corticosteroid levels and plasma adrenocorticotropic hormone (ACTH). Pulsed pump restored ultradian rhythmicity as demonstrated by five peaks of serum (all patients) and subcutaneous tissue cortisol (four patients). Morning subcutaneous cortisol and cortisone were higher in continuous and pulsed pump treatment than in oral therapy despite nearly similar serum cortisol levels in all treatment arms. ACTH was within the physiological range during pulsed pump treatment in all patients except for slightly elevated levels in the morning hours 04:00-08:00 h. During oral therapy, ACTH was very high in patients with Addison's disease and suppressed in patients with congenital adrenal hyperplasia. In conclusions, mimicking endogenous cortisol rhythmicity by ultradian subcutaneous infusion of cortisol is feasible. It was superior to both continuous pump and oral therapy in maintaining normal ACTH levels throughout the 24-h cycle. Our results demonstrate a low free cortisol bioavailability on thrice daily oral replacement therapy compared to both types of subcutaneous infusion.

Diabetic ketoacidosis with SGLT2 inhibitor use - a patient series. / Diabetisk ketoacidose ved bruk av SGLT2-hemmer ­ en pasientserie.

BACKGROUND: Inhibitors of sodium glucose cotransporter 2 (SGLT2 inhibitors) are increasingly being used to treat type 2 diabetes. Results from previous studies suggest a rising incidence of diabetic ketoacidosis with the use of this medication. MATERIAL AND METHOD: We performed a diagnosis search in the electronic patient records at Haukeland University Hospital for the period 1 January 2013-31 May 2021 with the aim of identifying patients with diabetic ketoacidosis who used SGLT2 inhibitors. A total of 806 patient records were reviewed. RESULTS: Twenty-one patients were identified. Thirteen had severe ketoacidosis, and ten had normal blood glucose levels. Probable triggering causes were found in 10 of the 21, with recent surgery being the most common (n = 6). Three of the patients were not tested for ketones, and 9 were not tested for antibodies to rule out type 1 diabetes. INTERPRETATION: The study showed that severe ketoacidosis occurs in patients with type 2 diabetes using SGLT2 inhibitors. It is important to be aware of this risk and the fact that ketoacidosis can occur without hyperglycaemia. Arterial blood gas and ketone tests must be performed to make the diagnosis.

Preclinical cardiac disease in women and men with primary aldosteronism.

PURPOSE: We tested the sex-specific associations between primary aldosteronism (PA), left ventricular (LV) hypertrophy and LV systolic myocardial function. MATERIAL AND METHODS: Conventional and speckle tracking echocardiography was performed in 109 patients with PA and 89 controls with essential hypertension (EH). LV hypertrophy was identified if LV mass index exceeded 47.0 g/m2.7 in women and 50.0 g/m2.7 in men. LV systolic myocardial function was assessed by global longitudinal strain (GLS) and midwall shortening. RESULTS: PA patients had higher prevalence of LV hypertrophy (52 vs. 21%, p < 0.001) than EH patients in both sexes, while GLS did not differ by sex or hypertension aetiology. In multivariable analyses, presence of LV hypertrophy was associated with PA and obesity in both sexes, while lower systolic myocardial function, whether measured by GLS or midwall shortening, was not associated with PA, but primarily with higher body mass index and LV mass index, respectively, in both sexes (all p < 0.05). CONCLUSION: Having PA was associated with higher prevalence of LV hypertrophy both in women and men, compared to EH. PA was not associated with LV systolic myocardial function in either sex.

Classic congenital adrenal hyperplasia. / Klassisk medfødt binyrebarkhyperplasi.

Congenital adrenal hyperplasia is attributed to inherited enzyme defects in the adrenal cortex. The classical form results in reduced production of cortisol and aldosterone, accompanied by an increase in production of adrenal cortical androgens. This causes virilisation in girls, adrenocortical failure and early puberty in both sexes. This article describes the genetics, clinical picture, diagnostics and treatment.

[Impaired sensitivity to thyroid hormone]. / Nedsatt følsomhet for thyreoideahormon.

BACKGROUND: In conditions with impaired sensitivity to thyroid hormone, reduced effect of thyroid hormone is attributable to various defects. The purpose of this article is to give an overview of these conditions, as well as to provide updated knowledge on impaired sensitivity to thyroid hormone, also known as thyroid hormone resistance, with mutations in thyroid hormone receptor ß (TRß). MATERIAL AND METHOD: This article is based on a selection of English-language articles, and Norwegian original and review articles found in PubMed, and the authors' own experiences with this patient group. RESULTS: Thyroid hormone resistance has long been a recognised cause of the reduced effect of thyroid hormone. Several other conditions that involve impaired sensitivity to thyroid hormone have been described in recent decades, and mutations have been identified in genes that code for thyroid hormone receptor α (TRα), a cell membrane transporter, as well as in the deiodinases that metabolise thyroxine (T4) to the bioactive form triiodothyronine (T3). The conditions vary in terms of their clinical picture and biochemical profile. INTERPRETATION: Based on clinical and biochemical findings, thyroid hormone resistance may be erroneously interpreted as hyperthyroidism. In patients with thyroid hormone resistance, the condition may be exacerbated if it is treated as hyperthyroidism. It is therefore essential to recognise the conditions and their differential diagnoses.

Circadian hormone profiles and insulin sensitivity in patients with Addison's disease: a comparison of continuous subcutaneous hydrocortisone infusion with conventional glucocorticoid replacement therapy.

CONTEXT: Conventional glucocorticoid replacement therapy in patients with Addison's disease (AD) is unphysiological with possible adverse effects on mortality, morbidity and quality of life. The diurnal cortisol profile can likely be restored by continuous subcutaneous hydrocortisone infusion (CSHI). OBJECTIVE: The aim of this study was to compare circadian hormone rhythms and insulin sensitivity in conventional thrice-daily regimen of glucocorticoid replacement therapy with CSHI treatment in patients with AD. DESIGN AND SETTING: An open, randomized, two-period, 12-week crossover multicentre trial in Norway and Sweden. PATIENTS: Ten Norwegian patients were admitted for 24-h sampling of hormone profiles. Fifteen Swedish patients underwent euglycaemic-hyperinsulinaemic clamp. INTERVENTION: Thrice-daily regimen of oral hydrocortisone (OHC) and CSHI treatment. MAIN OUTCOME MEASURE: We measured the circadian rhythm of cortisol, adrenocorticotropic hormone (ACTH), growth hormone (GH), insulin-like growth factor-1, (IGF-1), IGF-binding protein-3 (IGFBP-3), glucose, insulin and triglycerides during OHC and CSHI treatment. Euglycaemic-hyperinsulinaemic clamp was used to assess insulin sensitivity. RESULTS: Continuous subcutaneous hydrocortisone infusion provided a more physiological circadian cortisol curve including a late-night cortisol surge. ACTH levels showed a near normal circadian variation for CSHI. CSHI prevented a continuous decrease in glucose during the night. No difference in insulin sensitivity was observed between the two treatment arms. CONCLUSION: Continuous subcutaneous hydrocortisone infusion replacement re-established a circadian cortisol rhythm and normalized the ACTH levels. Patients with CSHI replacement had a more stable night-time glucose level compared with OHC without compromising insulin sensitivity. Thus, restoring night-time cortisol levels might be advantageous for patients with AD.

High cardiac background activity limits 99mTc-MIBI radioguided surgery in aortopulmonary window parathyroid adenomas.

BACKGROUND: Radioguided surgery using 99m-Technetium-methoxyisobutylisonitrile (99mTc-MIBI) has been recommended for the surgical treatment of mediastinal parathyroid adenomas. However, high myocardial 99mTc-MIBI uptake may limit the feasibility of radioguided surgery in aortopulmonary window parathyroid adenoma. CASE PRESENTATION: Two female patients aged 72 (#1) and 79 years (#2) with primary hyperparathyroidism caused by parathyroid adenomas in the aortopulmonary window were operated by transsternal radioguided surgery. After intravenous injection of 370 MBq 99mTc-MIBI at start of surgery, the maximum radioactive intensity (as counts per second) was measured over several body regions using a gamma probe before and after removal of the parathyroid adenoma. Relative radioactivity was calculated in relation to the measured ex vivo radioactivity of the adenoma, which was set to 1.0. Both patients were cured by uneventful removal of aortopulmonary window parathyroid adenomas of 4400 (#1) and 985 mg (#2). Biochemical cure was documented by intraoperative measurement of parathyroid hormone as well as follow-up examination. Ex vivo radioactivity over the parathyroid adenomas was 196 (#1) and 855 counts per second (#2). Before parathyroidectomy, relative radioactivity over the aortopulmonary window versus the heart was found at 1.3 versus 2.6 (#1) and 1.8 versus 4.8 (#2). After removal of the adenomas, radioactivity within the aortopulmonary window was only slightly reduced. CONCLUSION: High myocardial uptake of 99mTc-MIBI limits the feasibility of radioguided surgery in aortopulmonary parathyroid adenoma.

Prevalence, Risk Factors, and Clinical and Biochemical Characteristics of Alemtuzumab-Induced Graves Disease.

OBJECTIVE: Atypical Graves disease (GD) is a common complication in multiple sclerosis (MS) patients treated with alemtuzumab. We present epidemiological, clinical, and biochemical characteristics of alemtuzumab-induced GD. METHODS: Retrospective follow-up study of MS patients treated with alemtuzumab from 2014 to 2020, including clinical course of GD, pregnancy outcome, and thyroid eye disease (TED). RESULTS: We enrolled 183 of 203 patients (90%, 68% women) treated with alemtuzumab at 4 hospitals in Norway. Seventy-five (41%) developed thyroid dysfunction, of whom 58 (77%) had GD. Median time from the first dose of alemtuzumab to GD diagnosis was 25 months (range, 0-64). Twenty-four of 58 GD patients (41%) had alternating phases of hyper- and hypothyroidism. Thyrotropin receptor antibodies became undetectable in 23 of 58 (40%) and they could discontinue antithyroid drug treatment after a median of 22 (range, 2-58) months. Conversely, 26 (44%) had active disease during a median follow-up of 39 months (range, 11-72). Two patients (3%) received definitive treatment with radioiodine, 6 (10%) with thyroidectomy. Nine developed TED (16%), 7 had mild and 2 moderate to severe disease. Four patients completed pregnancy, all without maternal or fetal complications. Patients who developed GD had a lower frequency of new MS relapses and MRI lesions than those without. CONCLUSION: GD is a very common complication of alemtuzumab treatment and is characterized by alternating hyper- and hypothyroidism. Both remission rates and the prevalence of TED were lower than those reported for conventional GD. Pregnancies were uncomplicated and GD was associated with a lower risk of subsequent MS activity.

Outcomes of Patients With Graves Disease 25 Years After Initiating Antithyroid Drug Therapy.

CONTEXT: Graves disease (GD) is a leading cause of hyperthyroidism. Detailed investigations and predictors of long-term outcomes are missing. OBJECTIVE: This work aimed to investigate the outcomes in GD 25 years after initiating antithyroid drug treatment, including disease course, clinical and biochemical predictors of relapse, and quality of life. METHODS: A retrospective follow-up was conducted of GD patients that participated in a randomized trial from 1997 to 2001. Demographic and clinical data were obtained from medical records and questionnaires. Biobank samples were analyzed for inflammatory biomarkers and compared with age- and sex-matched healthy individuals. RESULTS: We included 83% (182/218) of the patients from the original study. At the end of follow-up, normal thyroid function was achieved in 34%. The remaining had either active disease (1%), spontaneous hypothyroidism (13%), or had undergone ablative treatment with radioiodine (40%) or thyroidectomy (13%). Age younger than or equal to 40 years, thyroid eye disease (TED), smoking, and elevated levels of interleukin 6 and tumor necrosis factor receptor superfamily member 9 (TNFRS9) increased the risk of relapsing disease (odds ratio 3.22; 2.26; 2.21; 1.99; 2.36). At the end of treatment, CD40 was lower in patients who maintained normal thyroid function (P = .04). At the end of follow-up, 47% had one or more autoimmune diseases, including vitamin B12 deficiency (26%) and rheumatoid arthritis (5%). GD patients who developed hypothyroidism had reduced quality of life. CONCLUSION: Careful lifelong monitoring is indicated to detect recurrence, hypothyroidism, and other autoimmune diseases. Long-term ATD treatment emerges as a beneficial first-line treatment option, especially in patients with young age at onset or presence of TED.

Assessing Lateralization Index of Adrenal Venous Sampling for Surgical Indication in Primary Aldosteronism.

BACKGROUND: Clinical practice guidelines recommend the Lateralization Index (LI) as the standard for determining surgical eligibility in primary aldosteronism (PA). Our goal was to identify the optimal LI cut-offs in adrenal venous sampling (AVS) for diagnosing PA that is amenable to surgical cure. METHODS: We conducted a retrospective international cohort study across 16 institutions in 11 countries, including 1,550 patients with PA who underwent AVS, with and/or without ACTH stimulation. The establishment of optimal cut-offs was informed by a survey of 82 PA patients in Japan, aimed at determining the LI cut-off aligned with patient expectations for a surgical cure rate. RESULTS: The survey revealed that a median cure rate expectation of 80% would motivate PA patients towards undergoing adrenalectomy. The optimal LI cut-offs achieving an adjusted positive predictive value (PPV) of 80% were identified as 3.8 for unstimulated AVS and 3.4 for ACTH-stimulated AVS. Furthermore, a contralateral ratio of less than 0.4 and the detection of an adrenal nodule on CT imaging were identified as independent predictors of surgically curable PA. Incorporating these factors with the optimal LI cut-offs, the adjusted PPV increased to 96.6% for unstimulated AVS and 89.6% for ACTH-stimulated AVS. No clear differences in predictive ability between unstimulated and ACTH-stimulated LI were found. CONCLUSIONS AND RELEVANCE: The present study clarified the optimal LI cut-offs for without and with ACTH stimulation. The presence of contralateral suppression and adrenal nodule on CT imaging seems to provide additional available information besides LI for surgical indication.

Substantial changes in inflammatory and cardiovascular biomarkers in patients with autonomous cortisol secretion.

OBJECTIVE: To map inflammatory biomarkers in patients with autonomous cortisol secretion (ACS) and overt Cushing syndrome (CS). METHOD: Observational study including serum from prospectively included patients with ACS (n = 63), adrenal CS (n = 2), pituitary CS (n = 8), and healthy subjects (n = 120). Serum samples were analysed for 92 inflammatory biomarkers using proximity extension assay (OLINK). RESULTS: Combined, the ACS and CS patients displayed significant differences in levels of 49/92 inflammatory biomarkers (46 increased/3 decreased) compared with healthy controls. No differences in biomarker levels were found between ACS and overt CS, and none of the biomarkers correlated with the degree of hypercortisolism. Postoperative samples were available for 17 patients, median 24 months (range 6-40) after surgery and biochemical curation. There was no significant normalization of the biomarkers postoperatively. CONCLUSION: There was a systemic rise in inflammatory biomarkers in patients with ACS and CS, not correlated to the degree of hypercortisolism. These biomarkers were not normalized following biochemical cure.

Systemic Activation of the Kynurenine Pathway in Graves Disease With and Without Ophthalmopathy.

CONTEXT: Graves disease (GD) is one of the most common autoimmune disorders. Recent literature has shown an immune response involving several different inflammatory related proteins in these patients. OBJECTIVE: This work aimed to characterize the kynurenine pathway, activated during interferon-γ (IFN-γ)-mediated inflammation and cellular (T-helper type 1 [Th1] type) immunity, in GD patients with and without thyroid eye disease (TED). METHODS: We analyzed 34 biomarkers by mass spectrometry in serum samples from 100 patients with GD (36 with TED) and 100 matched healthy controls. The analytes included 10 metabolites and 3 indices from the kynurenine pathway, 6 microbiota-derived metabolites, 10 B-vitamers, and 5 serum proteins reflecting inflammation and kidney function. RESULTS: GD patients showed significantly elevated levels of 7 biomarkers compared with healthy controls (omega squared [ω2] > 0.06; P < .01). Of these 7, the 6 biomarkers with the strongest effect size were all components of the kynurenine pathway. Factor analysis showed that biomarkers related to cellular immunity and the Th1 responses (3-hydroxykynurenine, kynurenine, and quinolinic acid with the highest loading) were most strongly associated with GD. Further, a factor mainly reflecting acute phase response (C-reactive protein and serum amyloid A) showed weaker association with GD by factor analysis. There were no differences in biomarker levels between GD patients with and without TED. CONCLUSION: This study supports activation of IFN-γ inflammation and Th1 cellular immunity in GD, but also a contribution of acute-phase reactants. Our finding of no difference in systemic activation of the kynurenine pathway in GD patients with and without TED implies that the local Th1 immune response in the orbit is not reflected systemically.

Persistent cardiac organ damage in surgically and medically treated primary aldosteronism.

OBJECTIVE: We compared persistent cardiac organ damage in patients treated surgically or medically for primary aldosteronism. METHODS: Eighty-four patients (age 57 ±â€Š11 years, 27% women) with primary aldosteronism underwent echocardiography at time of diagnosis and after one year of treatment (49% adrenalectomy, 51% medical treatment). Persistent cardiac organ damage was defined as presence of left ventricle (LV) hypertrophy, low LV midwall shortening, global longitudinal strain and/or enlarged left atrium both at baseline and at follow-up. RESULTS: At one year, a significant regression of LV hypertrophy was observed in surgically (44 vs. 22%, P  = 0.039), but not in medically treated patients (60 vs. 51%, P = 0.206). The prevalence of enlarged left atrium was reduced in both groups (both P < 0.001), whereas systolic myocardial function remained unchanged. In multivariable logistic regression analysis, medical treatment [odds ratio (OR) 4.88 (95% confidence interval (CI) 1.26-18.88)] was a strong predictor of persistent LV hypertrophy independent of higher BMI [OR 1.20 (95% CI 1.04-1.38)] and presence of diabetes [OR 6.48 (95% CI 1.20-34.83), all P < 0.05]. Persistently low midwall shortening was associated with suppressed plasma renin after one year [OR 6.11 (95% CI 1.39-26.7)] and lower renal function [OR 0.96 (95% CI 0.94-0.99), both P < 0.05]. The strongest predictor of persistently low global longitudinal strain was higher HbA1c [OR 2.37 (95% CI 1.12-5.02), P = 0.024]. CONCLUSION: Persistent cardiac organ damage was more common in the medical treatment group and associated with incomplete aldosterone blockade, impaired renal function and presence of metabolic comorbidities.http://links.lww.com/HJH/B925.

Novel inflammatory biomarkers in thyroid eye disease.

Purpose: The aim of this study is to identify biochemical inflammatory markers predicting the presence or risk of developing thyroid eye disease (TED) in patients with Graves' disease (GD). Methods: Patients with GD (n = 100, 77 females) were included from the National Norwegian Registry of Organ-Specific Diseases. Serum samples were analysed for 92 different inflammatory biomarkers using the proximity extension assay. Biomarker levels were compared between groups of patients with and without TED and healthy subjects (HS) (n = 120). Results: TED was found in 36 of 100 GD patients. Significant (P < 0.05) differences in the levels of 52 inflammatory biomarkers were found when GD patients and HS were compared (42 elevated and 10 decreased). Out of the 42 elevated biomarkers, a significantly higher serum level of interleukin-6 (IL6) (P = 0.022) and macrophage colony-stimulating factor (CSF1) (P = 0.015) were found in patients with TED compared to patients without TED. Patients with severe TED also had significantly elevated levels of Fms-related tyrosine kinase 3 ligand (FLT3LG) (P = 0.009). Furthermore, fibroblast growth factor 21 (FGF21) was significantly increased (P = 0.008) in patients with GD who had no signs of TED at baseline but developed TED later. Conclusion: We demonstrate an immunologic fingerprint of GD, as serum levels of several inflammation-related proteins were elevated, while others were decreased. Distinctly increased levels of IL6, CSF1, FLT3LG, and FGF21 were observed in TED, suggesting that these inflammatory proteins could be important in the pathogenesis, and therefore potential new biomarkers for clinical use.

Genome-wide copy number variation (CNV) in patients with autoimmune Addison's disease.

BACKGROUND: Addison's disease (AD) is caused by an autoimmune destruction of the adrenal cortex. The pathogenesis is multi-factorial, involving genetic components and hitherto unknown environmental factors. The aim of the present study was to investigate if gene dosage in the form of copy number variation (CNV) could add to the repertoire of genetic susceptibility to autoimmune AD. METHODS: A genome-wide study using the Affymetrix GeneChip® Genome-Wide Human SNP Array 6.0 was conducted in 26 patients with AD. CNVs in selected genes were further investigated in a larger material of patients with autoimmune AD (n = 352) and healthy controls (n = 353) by duplex Taqman real-time polymerase chain reaction assays. RESULTS: We found that low copy number of UGT2B28 was significantly more frequent in AD patients compared to controls; conversely high copy number of ADAM3A was associated with AD. CONCLUSIONS: We have identified two novel CNV associations to ADAM3A and UGT2B28 in AD. The mechanism by which this susceptibility is conferred is at present unclear, but may involve steroid inactivation (UGT2B28) and T cell maturation (ADAM3A). Characterization of these proteins may unravel novel information on the pathogenesis of autoimmunity.

High frequency of adrenal myelolipomas and testicular adrenal rest tumours in adult Norwegian patients with classical congenital adrenal hyperplasia because of 21-hydroxylase deficiency.

BACKGROUND: Increased frequencies of adrenal tumours and testicular adrenal rest tumours (TART) have been reported in patients with 21-hydroxylase deficiency (21OHD). OBJECTIVE: Patients, methods and design From a cross-sectional population-based study of 101 adult Norwegian patients with 21OHD, sixty-two participated in this study (23 men, 39 women; age range 18-75); thirty-two were salt wasting (SW) and 30 simple virilizing (SV); they were assessed with adrenal computed tomography (CT), testicular ultrasound and hormone measurement in the morning after overnight medication fast. RESULTS: Nine adrenal tumours were detected in seven (11%) patients (bilateral in 2); four were myelolipomas and one a phaeochromocytoma. Seventeen (27%) had normal adrenal size, whereas 36 (58%) had persisting hyperplasia, and seven (11%) adrenal hypoplasia. Abnormal adrenals were more common in SW than in SV. TART occurred exclusively in SW and was present in seven (57%) of these men. Testicular volumes were small compared with normative data. Morning ACTH and 17-hydroxyprogesterone levels correlated positively with adrenal dimensions and frequency of TART. CONCLUSION: In this unselected population of patients with classical 21OHD, we found high frequencies of adrenal tumours, particularly myelolipomas, and of hyperplasia and hypoplasia, and TART in SW. It is important that physicians are aware that benign adrenal and testicular tumours occur frequently in 21OHD. Furthermore, these findings may reflect inappropriate glucocorticoid therapy, making a case for the advancement of novel physiological treatment modalities.

Potential Transcriptional Biomarkers to Guide Glucocorticoid Replacement in Autoimmune Addison's Disease.

BACKGROUND: No reliable biomarkers exist to guide glucocorticoid (GC) replacement treatment in autoimmune Addison's disease (AAD), leading to overtreatment with alarming and persistent side effects or undertreatment, which could be fatal. OBJECTIVE: To explore changes in gene expression following different GC replacement doses as a means of identifying candidate transcriptional biomarkers to guide GC replacement in AAD. METHODS: Step 1: Global microarray expression analysis on RNA from whole blood before and after intravenous infusion of 100 mg hydrocortisone (HC) in 10 patients with AAD. In 3 of the most highly upregulated genes, we performed real-time PCR (rt-PCR) to compare gene expression levels before and 3, 4, and 6 hours after the HC infusion. Step 2: Rt-PCR to compare expression levels of 93 GC-regulated genes in normal versus very low morning cortisol levels in 27 patients with AAD. RESULTS: Step 1: Two hours after infusion of 100 mg HC, there was a marked increase in FKBP5, MMP9, and DSIPI expression levels. MMP9 and DSIPI expression levels correlated with serum cortisol. Step 2: Expression levels of CEBPB, DDIT4, FKBP5, DSIPI, and VDR were increased and levels of ADARB1, ARIDB5, and POU2F1 decreased in normal versus very low morning cortisol. Normal serum cortisol levels positively correlated with DSIPI, DDIT4, and FKBP5 expression. CONCLUSIONS: We introduce gene expression as a novel approach to guide GC replacement in AAD. We suggest that gene expression of DSIPI, DDIT4, and FKBP5 are particularly promising candidate biomarkers of GC replacement, followed by MMP9, CEBPB, VDR, ADARB1, ARID5B, and POU2F1.

Diagnostic testing of autonomous cortisol secretion in adrenal incidentalomas.

OBJECTIVE: Autonomous cortisol secretion (ACS) is a condition with ACTH-independent cortisol overproduction from adrenal incidentalomas (AI) or adrenal hyperplasia. The hypercortisolism is often mild, and most patients lack typical clinical features of overt Cushing's syndrome (CS). ACS is not well defined and diagnostic tests lack validation. METHODS: Retrospective study of 165 patients with AI evaluated clinically and by assay of morning plasma ACTH, late-night saliva cortisol, serum DHEA sulphate (DHEAS), 24-h urine-free cortisol, and cortisol after dexamethasone suppression. RESULTS: Patients with AI (n = 165) were diagnosed as non-functioning incidentalomas (NFI) (n = 82) or ACS (n = 83) according to current European guidelines. Late-night saliva cortisol discriminated poorly between NFI and ACS, showing a high rate of false-positive (23/63) and false-negative (38/69) results. The conventional low-dose dexamethasone suppression test (LDDST) did not improve the diagnostic specificity, compared with the 1 mg overnight DST. Receiver operating characteristic curve analysis of DHEAS in the two cohorts demonstrated an area under the curve of 0.76 (P < 0.01) with a sensitivity for ACS of 58% and a specificity of 80% using the recommended cutoff at 1.04 µmol/L (40 µg/dL). CONCLUSION: We here demonstrate in a large retrospective cohort of incidentaloma patients, that neither DHEAS, late-night saliva cortisol nor 24-h urine free cortisol are useful to discriminate between non-functioning adrenal incidentalomas and ACS. The conventional LDDST do not add further information compared with the 1 mg overnight DST. Alternative biomarkers are needed to improve the diagnostic workup of ACS.