Next-generation sequencing identifies novel mitochondrial variants in pituitary adenomas.

PURPOSE: Disrupted mitochondrial functions and genetic variants of mitochondrial DNA (mtDNA) have been observed in different human neoplasms. Next-generation sequencing (NGS) can be used to detect even low heteroplasmy-level mtDNA variants. We aimed to investigate the mitochondrial genome in pituitary adenomas by NGS. METHODS: We analysed 11 growth hormone producing and 33 non-functioning [22 gonadotroph and 11 hormone immunonegative] pituitary adenomas using VariantPro™ Mitochondrion Panel on Illumina MiSeq instrument. Revised Cambridge Reference Sequence (rCRS) of the mtDNA was used as reference. Heteroplasmy was determined using a 3% cutoff. RESULTS: 496 variants were identified in pituitary adenomas with overall low level of heteroplasmy (7.22%). On average, 35 variants were detected per sample. Samples harbouring the highest number of variants had the highest Ki-67 indices independently of histological subtypes. We identified eight variants (A11251G, T4216C, T16126C, C15452A, T14798C, A188G, G185A, and T16093C) with different prevalences among different histological groups. T16189C was found in 40% of non-recurrent adenomas, while it was not present in the recurrent ones. T14798C and T4216C were confirmed by Sanger sequencing in all 44 samples. 100% concordance was found between NGS and Sanger method. CONCLUSIONS: NGS is a reliable method for investigating mitochondrial genome and heteroplasmy in pituitary adenomas. Out of the 496 detected variants, 414 have not been previously reported in pituitary adenoma. The high number of mtDNA variants may contribute to adenoma genesis, and some variants (i.e., T16189C) might associate with benign behaviour.

Diagnostic performance of a newly developed salivary cortisol and cortisone measurement using an LC-MS/MS method with simple and rapid sample preparation.

BACKGROUND: Late-night salivary cortisol level is one of the first-line tests recommended by the Endocrine Society for the diagnosis of endogenous hypercortisolism. Most routine laboratories measure cortisol levels using immunoassay tests which fail to determine low cortisol levels accurately due to the numerous interfering substances. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with simple and rapid sample preparation was developed for the simultaneous measurement of cortisol and cortisone and its performance in the diagnosis of endogenous hypercortisolism was evaluated. METHODS: 324 late-night saliva samples were analyzed from which 272 samples were derived from patients with a suspected diagnosis of endogenous hypercortisolism. Salivary cortisol levels were assayed using an electrochemiluminescent immunoassay (ECLIA, Cortisol II, Roche), and simultaneous measurement of cortisol and cortisone was performed using an LC-MS/MS method. RESULTS: A strong correlation between cortisol results measured using ECLIA and LC-MS/MS (r 2 = 0.892) was demonstrated. Receiver operating characteristics (ROC) analysis showed good diagnostic performance of cortisol and cortisone levels assayed using LC-MS/MS method and for cortisol measured using ECLIA. CONCLUSIONS: Late-night salivary cortisol and cortisone are useful parameters for the diagnosis of hypercortisolism. Using samples obtained from patients where the diagnosis of hypercortisolism is extremely challenging cut-off values for midnight salivary cortisol and cortisone measured by LC-MS/MS method were established.

Bone turnover in patients with endogenous Cushing's syndrome before and after successful treatment.

UNLABELLED: We investigated bone turnover and its restoration in a large number of patients in the active phase and after cure of endogenous Cushing's syndrome. Furthermore, the usefulness of serum osteocalcin and collagen breakdown products as potential markers of active Cushing's syndrome was also evaluated. INTRODUCTION: Suppressed bone formation is one of the most characteristic features of Cushing's syndrome (CS). Despite numerous previous reports, many aspects of the disturbed bone metabolism of these patients are unexplored. In this study, we investigated the time course of bone marker changes after the cure of CS as well as correlations between bone markers and serum cortisol concentrations. METHODS: Eighty-seven patients with CS were studied. Patients were followed up to 48 months after surgical cure. Serum osteocalcin (OC) and collagen breakdown products (CTX) were measured with immunochemiluminescence method and compared to the results of 161 healthy controls. RESULTS: OC showed a negative, while CTX displayed a positive correlation with serum cortisol. Patients with diabetes mellitus and myopathy had significantly lower serum OC levels compared to those without these complications. The area under the curve of OC obtained by receiver-operating characteristics analysis for the discrimination of patients with CS from healthy controls was 0.9227. Postoperative OC increased rapidly from the first few days or weeks reaching its maximum at the sixth month and remained stable after the 24th postoperative month. CONCLUSIONS: Our study demonstrated significant correlations between serum cortisol and both bone formation and resorption markers in the active phase of CS. We propose that OC may serve as a sensitive biologic marker of glucocorticoid activity in endogenous CS during its active phase and it may reflect the clinical cure of the disease.

Clinical and biochemical features of sporadic and hereditary phaeochromocytomas: an analysis of 41 cases investigated in a single endocrine centre.

The aims of this study were to estimate the prevalence of phaeochromocytomas among adrenal tumours and to analyse the clinical and biochemical features of sporadic and hereditary tumours. Our series of 609 adrenal tumours evaluated between January 1995 and July 2003 was reviewed. Catecholamine content in phaeochromocytoma tissues was also determined and correlated with clinical behaviour and biochemical parameters of patients. Forty-one (6.7%) of the 609 patients had phaeochromocytomas, of which 28 were sporadic (25 benign and three malignant) and 13 (all benign) were associated with hereditary diseases (multiple endocrine neoplasia type 2A in seven cases from four unrelated families carrying mutations of the RET gene, von Hippel-Lindau disease in two unrelated cases with mutations of the VHL gene, and type 1 neurofibromatosis in four unrelated cases). Bilateral tumours were found in three patients with hereditary syndromes and in one sporadic case. Tumour diameter was slightly but not significantly greater in patients with hereditary than in those with sporadic tumours. Systolic but not diastolic blood pressure was significantly higher in patients with sporadic compared with those with hereditary tumours, but comparison of other clinical data and biochemical parameters indicated an absence of significant differences in the mean age, presenting symptoms, heart rate, or fasting serum glucose levels. Tissue catecholamine content measured in 8 sporadic and 5 hereditary phaeochromocytomas was highly variable and it failed to show significant differences between hereditary and sporadic tumours. These results indicate a high proportion of hereditary diseases among patients with phaeochromocytomas. Genetic and clinical testing for hereditary diseases may be of great help to offer an appropriate treatment, follow-up and family screening for these patients.

[Endocrinologic complications of neurofibromatosis type 1]. / Neurofibromatosis 1. típusának endokrinológiai szövódményei.

Neurofibromatosis type 1 is the most common autosomal dominant inheritable disease, which is often associated with secondary forms of hypertension and with tumors of neuroectodermal origin. The authors present the results of evaluation of 60 members of 3 families. Of the 60 family members, 13 subjects had symptoms of neurofibromatosis type 1 disease, of which 7 subjects were evaluated. The case histories of patients are discussed: (1) An incidentally discovered adrenal tumor was proved to be a pheochromocytoma. (2) Because of complaints similar to thyrotoxicosis, thyrostatic drugs were administered for years without effect and, finally, an adrenal phaeochromocytoma was diagnosed after the presence of neurofibromatosis was established. (3) Preeclamptic pregnancy of a young primigravida complicated with severe HELLP syndrome (hemolysis-elevated liver enzymes-low platelet count) led to thorough evaluation which revealed renal artery stenosis. In this patient, percutaneous renal artery angioplasty resulted in a complete cessation of hypertension. (4) Glucocortocoid replacement therapy in a patient with neurofibromatosis type 1 resulted in a complete normalization of both secondary adrenal insufficiency and a previously unexplained iron-refractor iron-deficient anemia. The case histories of the patients demonstrate a lack of in-depth knowledge of neurofibromatosis in clinical practice. A regular follow-up of neurofibromatosis patients is suggested in specialized health centers.

[Effects of growth hormone replacement therapy in adults with severe growth hormone deficiency]. / Növekedési hormonnal történó hormonpótló kezelés hatásai súlyos növekedési hormonhiányos felnóttekben.

UNLABELLED: The aim of the study was to analyse the effects of GH replacement therapy (1 year duration) on body composition, carbohydrate metabolism, thyroid hormone metabolism and bone mineral density in 8 adults with growth hormone deficiency (5 women, 3 men; mean age 40 years). Mean maintenance dose of GH was 1.5 IU/day-1.76 IU/day for women and 1.07 IU/day for men, respectively--determined according to individual patient requirements. Serum insulin-like growth factor-I standard deviation score increased from -5.4 to 0.0 (p < 0.001). There was a significant negative relationship between serum insulin-like growth factor-I standard deviation score at the start of therapy and the increase in this score (r = -0.85; p < 0.05). The waist:hip ratio decreased after 12 months by 0.039 (p < 0.05). The glycosylated hemoglobin increased (4.43 +/- 0.56% vs. 5.86 +/- 0.27; p < 0.05), and a negative correlation of the baseline glycosylated hemoglobin to the glycosylated hemoglobin increase was found (r = -0.88; p < 0.01). Both the free triiodothyronine and free triiodothyronine:free thyroxine ratio increased (3.09 +/- 0.22 vs. 4.17 +/- 0.40; p < 0.05, and 0.234 +/- 0.02 vs. 0.324 +/- 0.04; p < 0.01), and a positive relationship was observed between this ratio at the start of therapy and the increase in the ratio (r = 0.76, p < 0.05). The bone mineral density of lumbar spine and femoral neck expressed as z-score increased (-1.18 +/- 0.56 vs. -0.75 +/- 0.48; p < 0.01 and -0.06 +/- 0.60 vs. 0.43 +/- 0.43; p < 0.05), while the bone mineral density of forearm was unchanged. CONCLUSIONS: Growth hormone replacement leads to a decrease in visceral fat, modulates the thyroid hormone levels by increasing peripheral conversion of thyroxine to triiodothyronine and probably is a physiological regulator of peripheral thyroxine metabolism, slightly deteriorates the carbohydrate metabolism, and results in an increase of bone mineral density of lumbar spine and femoral neck.

Neurofibromatosis type 1 with pregnancy-associated renovascular hypertension and the syndrome of hemolysis, elevated liver enzymes, and low platelets.

The authors detected a case of neurofibromatosis type 1 complicated with preeclampsia and the syndrome of hemolysis, elevated liver enzymes, and low platelets, which developed during pregnancy. When the cause of the patient's hypertension was investigated after birth, renal stenosis of the right artery was detected, which was then successfully treated with percutaneous transluminal angioplasty.

Leptin inhibits cortisol and corticosterone secretion in pathologic human adrenocortical cells.

Regulation of adrenal corticosteroid secretion by leptin may involve interactions at multiple levels of the hypothalamic-pituitary-adrenal axis. To investigate the possible direct effects of leptin on corticosteroid secretion of human adrenocortical adenomas, cells from adrenocortical adenomas causing primary aldosteronism (n = 1) and Cushing's syndrome (n = 1), as well as cells from nonhyperfunctioning adrenocortical adenomas (n = 5) were isolated and incubated for 2 h with human recombinant leptin (1-1000 ng/ml) in the presence and absence of adrenocorticotrop hormone (ACTH), then cortisol, corticosterone and aldosterone concentrations in incubating media were determined using radioimmunoassays. It was found that leptin effectively and dose-dependently inhibited basal and ACTH-stimulated cortisol and corticosterone secretion in the three types of human adrenocortical adenoma cells. The inhibiting effect of basal corticosterone secretion was detectable in the presence of leptin concentration as low as 1 ng/ml, with decreases of corticosterone secretion to 34+/-4%, 57+/-11% and 79+/-9% in Cushing's syndrome, primary aldosteronism, and nonhyperfunctioning adrenocortical adenoma cells, respectively. The inhibition of basal cortisol secretion in the presence of low concentration of leptin was less prominent, but 10 ng/ml leptin significantly diminished basal cortisol secretion to 81+/-9% in adrenocortical adenoma cells from Cushing's syndrome, to 68+/-6% in adenoma cells from primary aldosteronism, and to 83+/-8% in cells from nonhyperfunctioning adenomas. The inhibition of ACTH-stimulated cortisol and corticosterone secretion by leptin was similar to those found in cells without ACTH stimulation. By contrast, leptin even at 1000 ng/ml concentration exerted no clear effect on basal and ACTH-stimulated aldosterone secretion in cells from primary aldosteronism and in those nonhyperfunctioning adenoma cells in which aldosterone secretion was detectable. These results indicate that leptin is a potent inhibitor of cortisol and corticosterone secretion in human adenomatous adrenocortical cells. The inhibition of these corticosteroids by leptin may represent a potentially important interaction that exists between leptin and the hypothalamic-pituitary-adrenal axis.

Effects of 24 months of growth hormone (GH) treatment on serum carboxylated and undercarboxylated osteocalcin levels in GH-deficient adults.

We studied the effect of growth hormone (GH) replacement on bone mineral density (BMD) and some parameters of bone metabolism, including undercarboxylated osteocalcin (ucOC), an independent predictive marker of fracture risk, which has not been previously determined or compared during GH treatment. Measurements were performed at baseline and after 6, 12, 18 and 24 months of the initiation of the GH therapy in 21 adult patients with GH deficiency. Significant increases were observed in BMD after 1 year at the lumbar spine and after 1.5 years at the femoral neck. Serum total OC and carboxylated (c) OC increased and reached the maximum at 6 months, but the values remained over the baseline at both 12 and 18 months. The ucOC:total OC ratio changed contrarily: it decreased at 6 months, then increased again and reached the baseline level during the next 18 months. Serum calcium (Ca), phosphate (P) and total alkaline phosphatase (ALP) levels increased after 6 months, thereafter the Ca and P values decreased, while the total ALP remained elevated until 12 months. Serum parathormone decreased at 12 months and increased again thereafter. GH replacement therapy is associated with improvement of ucOC, a marker of fracture risk, which in addition to the increase of BMD, might contribute to the beneficial effect of GH replacement therapy on bone metabolism.

Ovarian steroid cell tumor and a contralateral ovarian thecoma in a postmenopausal woman with severe hyperandrogenism.

A 49-year-old woman presented with rapidly progressing hirsutism, receding hairline, male-pattern baldness and deepening of voice, which had developed over the past 2 years. Hormonal evaluation showed a markedly elevated serum testosterone level (418 ng/dl) and no evidence of increased production of cortisol, dehydroepiandrosterone, dehydroepiadrosterone-sulfate, androstenedione, or 17-hydroxyprogesterone. Transvaginal ultrasound examination suggested the presence of a small mass within the left ovary, but all other radiological studies, including adrenal and ovarian computed tomography, magnetic resonance imaging, radio-labelled cholesterol scintigraphy and positron emission tomography, were negative. Subsequently, bilateral selective venous sampling showed a marked testosterone gradient in the right ovarian vein. Bilateral salpingo-oophorectomy was performed (the patient had had a previous vaginal hysterectomy), and histopathological examination revealed a 10-mm steroid cell tumor within the right ovary and a 15-mm thecal cell tumor within the left ovary. The postoperative serum testosterone level returned to normal and the patient showed a slow regression of clinical symptoms. The simultaneous occurrence of a virilizing ovarian steroid cell tumor and an apparently non-functioning thecoma within the contralateral ovary emphasizes the potential pitfalls that may exist in the preoperative evaluation of patients with markedly increased testosterone production.

Effect of single doses of dexamethasone and adrenocorticotrop hormone on serum bone markers in healthy subjects and in patients with adrenal incidentalomas and Cushing's syndrome.

The aim of the present study was to explore whether short-term changes in glucocorticoid activity which occur during dynamic testing of the pituitary adrenal axis with dexamethasone, ACTH, or metyrapone could have an effect on serum osteocalcin (OC) and beta-crosslaps (beta-CTx) concentrations in healthy subjects, in patients with adrenal incidentalomas and in those with Cushing's syndrome. The study included 40 healthy subjects (35 women and 5 men, age range 18-69 yr), 49 patients with adrenal incidentalomas (34 women and 15 men, age range 19-77 yr) and 8 patients with Cushing's syndrome (5 cortisol-producing adenomas and 3 pituitary-dependent Cushing's syndrome, 3 women and 5 men, age range 19-70 yr). Serum OC and beta-CTx concentrations were determined with electrochemoluminescent immunoassays at midnight, after an overnight fast between 08:00 and 09:00 h, after an overnight dexamethasone test (1 mg, orally) and after a single dose of metyrapone (30 mg/kg, orally). In healthy subjects and in patients with adrenal incidentalomas, serum bone marker concentrations were also measured after a single dose of ACTH injection (Cortrosyn depot, 1 mg im). Patients with Cushing's syndrome, but not those with adrenal incidentalomas, showed significantly lower serum OC at midnight (18.5+/-12 ng/ml, mean+/-SD) and between 08:00 and 09:00 h (17.7+/-9.6 ng/ml) compared to corresponding values obtained in healthy subjects (24.5+/-7.0 and 28.3+/-12.2 ng/ml, respectively). Serum OC concentrations were significantly decreased after a single dose of 1-mg dexamethasone in healthy subjects (from 28.3+/-12.2 to 21.8+/-9.5 ng/ml) and in patients with adrenal incidentalomas (from 29.8+/-15.9 to 24.1+/-14.1 ng/ml), whereas serum OC concentrations remained unchanged in patients with Cushing's syndrome. In addition, serum OC concentrations were even more markedly decreased after a single dose of ACTH injection in both healthy subjects (12.5+/-4.6 ng/ml) and in patients with adrenal incidentalomas (12.2+/-6.5 ng/ml). By contrast, metyrapone administration failed to induce significant changes in OC levels. There were no significant differences in beta-CTx concentrations between the three groups or after drug treatments. Thus, serum OC levels should be interpreted with caution when obtained during testing of the pituitary-adrenal axis with dexamethasone or ACTH.

Plasma and salivary 6beta-hydroxycortisol measurements for assessing adrenocortical activity in patients with adrenocortical adenomas.

The aim of this study was to examine and compare the potential usefulness of plasma and salivary 6beta-hydroxycortisol measurements for assessing adrenocortical activity in patients with adrenocortical adenomas. Plasma and salivary cortisol as well as 6beta-hydroxycortisol determinations were performed by radioimmunoassay after extraction with ethyl acetate followed by chromatographic separation using a modified paper chromatographic system. Samples were obtained from 36 control subjects and 37 patients with non-hyperfunctioning adrenocortical adenomas in the morning at 8 a.m. after a low-dose of dexamethasone and after stimulation with synthetic depot ACTH. Basal and post-dexamethasone hormone levels were also measured in plasma and salivary samples of 4 patients with Cushing's syndrome from adrenal adenomas. In the baseline state, patients with non-hyperfunctioning adrenocortical adenomas had significantly higher plasma and salivary 6beta-hydroxycortisol levels (mean+/-SE, 79.0+/-7 and 17.1+/-2.2 ng/dl, respectively) compared to those measured in controls (62.0+/-4 and 7.7+/-0.6 ng/dl, respectively), whereas baseline plasma and salivary cortisol levels (9.6+/-0.5 microg/dl and 342+/-39 ng/dl, respectively) were similar to those measured in the control group (9.9+/-0.4 microg/dl and 366+/-24 ng/dl, respectively). In all groups, the changes in plasma and salivary 6beta-hydroxycortisol concentrations after dexamethasone suppression and ACTH stimulation were similar to the changes in plasma and salivary cortisol levels, although the differing ratios of 6betaOHF to cortisol indicated potentially important variations in the induction of 6beta-hydroxylase activity between the three groups. In patients with Cushing's syndrome, baseline plasma and salivary 6beta-hydroxycortisol concentrations (754+/-444 and 104+/-88 ng/dl, respectively) were more markedly increased than plasma and salivary cortisol levels (24.8+/-6.7 microg/dl and 1100+/-184 ng/dl, respectively), and all remained non-suppressible after dexamethasone administration. These results suggests that plasma and salivary 6beta-hydroxycortisol determinations may precisely detect not only overt increases of cortisol secretion in patients with Cushing's syndrome but also mild glucocorticoid overproduction presumably present in patients with non-hyperfunctioning adrenocortical tumors.

The effect of gender and age on growth hormone replacement in growth hormone-deficient patients.

We analyzed the effect of growth hormone replacement therapy (36 months) analyzed at a dose adjusted to maintain serum insulin-like growth factor-I level between the median and the upper end of the age-related reference range on bone mineral density, body composition, and carbohydrate metabolism with respect to gender and age in 20 adult patients (9 women, 11 men, mean age: 43 years, range: 21-61 years). The lumbar and femoral T-score was increased after 12 and after 18 months of therapy respectively in men (p < 0.001 and p = 0.002), but did not changed significantly in women. The increase of femoral T-score was greater in young men (< or = 45 years, n = 6) than old men (> 45 years, n = 5, p < 0.001). Body fat was lower in men than in women after 6 months (p = 0.002). The waist/hip ratio only decreased in women (p = 0.044). The waist circumference decreased in both genders after 6 months of therapy (p < 0.001), but more markedly in females than in males (p < 0.05). The sum of skinfold thicknesses was reduced in males after 6 months of therapy (p < 0.001). Changes in body composition parameters measured were independent of age. The glycosylated hemoglobin increased without sex or age difference after 12 months of initiation of therapy (p < 0.001), but fasting glucose and insulin levels did not change during the therapy. Our results indicate that the effect of growth hormone replacement on bone mineral content in adults is age- and gender-dependent, gender dependent on body composition, but independent of age and gender on carbohydrate metabolism.