The effects of mitotane and 1α,25-dihydroxyvitamin D3 on Wnt/beta-catenin signaling in human adrenocortical carcinoma cells.

PURPOSE: Mitotane is the only chemotherapeutic agent available for the treatment of adrenocortical carcinoma (ACC), however, the anti-neoplastic efficacy is limited due to several side-effects in vivo. There is, therefore, a need of exploring for new anti-tumoral agents which can be used either alone or in combination with mitotane. The active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) acts as an anti-proliferative agent in human cancer by inhibiting the Wnt/beta-catenin pathway through the vitamin D receptor (VDR). The aim of this study was to study the effects of mitotane and 1α,25(OH)2D3, individually or in combination, in an in vitro model with H295R ACC cells, and to elucidate the molecular events behind their effects involving the Wnt/beta-catenin signaling. METHODS AND RESULTS: Multiple concentrations of mitotane and 1α,25(OH)2D3, individually or in combination, were tested on H295R cells for 24-96 h, and the effects analysed by MTT. A reduction in cell growth was observed in a dose/time-dependent manner for both mitotane and 1α,25(OH)2D3. In addition, a combination of clinically sub-therapeutic concentrations of mitotane with 1α,25(OH)2D3, had an additive anti-proliferative effect (Combination Index = 1.02). In a wound healing assay, individual treatments of both mitotane and 1α,25(OH)2D3 reduced the migration ability of H295R cells, with the effect further enhanced on combining both the agents. Western blotting and qRT-PCR analysis showed a modulation of the Wnt/beta-catenin and VDR signaling pathways. CONCLUSION: Our results show an additive effect of mitotane and 1α,25(OH)2D3 on the inhibition of H295R ACC cell growth and viability, and suggest that molecular mechanisms of their effects involve a functional link between VDR and Wnt/beta-catenin pathways.

Sleep disorders and cognitive dysfunction in acromegaly.

PURPOSE: In the general population, sleep disorders are associated with an increased risk of cognitive impairment. The prevalence of sleep disorders, such as sleep apnea, in acromegalic patients is higher than in the general population, and they may have additional risk of cognitive impairment due to acromegaly treatment and comorbidities. We aim to study the relationship between sleep disturbances and cognitive dysfunction in a group of acromegalic patients. METHODS: We studied 67 consecutive acromegalic patients. We performed a neurocognitive assessment and patients completed the Acromegaly Quality of Life Questionnaire (AcroQoL), Epworth Sleepiness Scale, and Pittsburgh Sleep Quality Index. RESULTS: Of the 67 acromegaly patients in the study, 38.8% were male and median age at the neurological examination was 56 (IQR 48, 65). Approximately 6-10% of patients had impaired cognitive assessment, depending on the test. In linear regression models adjusted for age, sex, BMI, disease duration, and disease activity, poorer sleep quality was associated with lower global cognitive z-score (B = -0.03, 95% CI -0.06, -0.002). Daytime somnolence was associated with poorer physical AcroQoL sub-score (B = -0.04, 95% CI -0.08, -0.002). Sleep quality was associated with poorer overall AcroQoL (B = -0.03, 95% CI -0.05, -0.006), physical AcroQoL (B = -0.04, 95% CI -0.07, -0.005), psychological AcroQoL (B = -0.02, 95% CI -0.04, -0.001), and social AcroQoL (B = -0.02, 95% CI -0.04, -0.0009). CONCLUSIONS: In acromegaly patients, we found robust evidence that poor sleep quality is associated with poorer quality of life, and some evidence that it is associated with poorer cognitive function.

Guidelines versus real life practice: the case of colonoscopy in acromegaly.

PURPOSE: The aim of this study is to investigate guideline application and colonoscopy findings in real-life practice in acromegaly. METHODS: We conducted a retrospective observational non-interventional and cross-sectional analysis on 146 patients with acromegaly (ACRO) referred to our clinic. We evaluated colonoscopy data, focusing on the correlation between colonoscopy findings and hormonal/metabolic values. RESULTS: The total number of colonoscopies performed in ACRO patients increased from 6 in the period 1990-1994 to 57 in the period 2010-2014. Colonoscopy procedures were performed according to guidelines in 25% of ACRO patients at diagnosis, 51% at follow-up and 11% globally (both at diagnosis and follow-up). Among the 146 ACRO patients, 68% were subjected to at least one colonoscopy and in 32% of the cases a polyp was detected during the procedure. The presence of polyps was significantly associated with mean levels of growth hormone (GH), insulin-like growth factor 1 (IGF-1), fasting glucose and insulin levels (p < 0.05). Polyps were detected in 48% of untreated patients and in 26% of patients under treatment for acromegaly (p = 0.04). The general risk of polyps and adenomatous polyps in ACRO patients was higher compared to the control population of Veneto Region, Italy (odds ratio 1.33 and 1.16, respectively). No cancerous polyps were detected in our analysis. CONCLUSION: In real-life practice, adherence to ACRO colonoscopy clinical guidelines was lower than expected. Among patients who underwent colonoscopy, the prevalence of colon polyps was higher for ACRO patients, suggesting the need for new strategies to ensure adherence to colonoscopy guidelines.

Ambulatory blood pressure monitoring-derived short-term blood pressure variability in primary hyperparathyroidism.

INTRODUCTION: Primary hyperparathyroidism is associated with a cluster of cardiovascular manifestations, including hypertension, leading to increased cardiovascular risk. PURPOSE: The aim of our study was to investigate the ambulatory blood pressure monitoring-derived short-term blood pressure variability in patients with primary hyperparathyroidism, in comparison with patients with essential hypertension and normotensive controls. METHODS: Twenty-five patients with primary hyperparathyroidism (7 normotensive,18 hypertensive) underwent ambulatory blood pressure monitoring at diagnosis, and fifteen out of them were re-evaluated after parathyroidectomy. Short-term-blood pressure variability was derived from ambulatory blood pressure monitoring and calculated as the following: 1) Standard Deviation of 24-h, day-time and night-time-BP; 2) the average of day-time and night-time-Standard Deviation, weighted for the duration of the day and night periods (24-h "weighted" Standard Deviation of BP); 3) average real variability, i.e., the average of the absolute differences between all consecutive BP measurements. RESULTS: Baseline data of normotensive and essential hypertension patients were matched for age, sex, BMI and 24-h ambulatory blood pressure monitoring values with normotensive and hypertensive-primary hyperparathyroidism patients, respectively. Normotensive-primary hyperparathyroidism patients showed a 24-h weighted Standard Deviation (P < 0.01) and average real variability (P < 0.05) of systolic blood pressure higher than that of 12 normotensive controls. 24-h average real variability of systolic BP, as well as serum calcium and parathyroid hormone levels, were reduced in operated patients (P < 0.001). A positive correlation of serum calcium and parathyroid hormone with 24-h-average real variability of systolic BP was observed in the entire primary hyperparathyroidism patients group (P = 0.04, P = 0.02; respectively). CONCLUSION: Systolic blood pressure variability is increased in normotensive patients with primary hyperparathyroidism and is reduced by parathyroidectomy, and may potentially represent an additional cardiovascular risk factor in this disease.

ARMC5 mutation analysis in patients with primary aldosteronism and bilateral adrenal lesions.

Idiopathic hyperaldosteronism (IHA) due to bilateral adrenal hyperplasia is the most common subtype of primary aldosteronism (PA). The pathogenesis of IHA is still unknown, but the bilateral disease suggests a potential predisposing genetic alteration. Heterozygous germline mutations of armadillo repeat containing 5 (ARMC5) have been shown to be associated with hypercortisolism due to sporadic primary bilateral macronodular adrenal hyperplasia and are also observed in African-American PA patients. We investigated the presence of germline ARMC5 mutations in a group of PA patients who had bilateral computed tomography-detectable adrenal alterations. We sequenced the entire coding region of ARMC5 and all intron/exon boundaries in 39 patients (37 Caucasians and 2 black Africans) with confirmed PA (8 unilateral, 27 bilateral and 4 undetermined subtype) and bilateral adrenal lesions. We identified 11 common variants, 5 rare variants with a minor allele frequency <1% and 2 new variants not previously reported in public databases. We did not detect by in silico analysis any ARMC5 sequence variations that were predicted to alter protein function. In conclusion, ARMC5 mutations are not present in a fairly large series of Caucasian patients with PA associated to bilateral adrenal disease. Further studies are required to definitively clarify the role of ARMC5 in the pathogenesis of adrenal nodules and aldosterone excess in patients with PA.

Heart rate variability is reduced in acromegaly patients and improved by treatment with somatostatin analogues.

BACKGROUND: Cardiovascular complications, including arrhythmias and cardiac sudden death, are the most common causes of enhanced mortality in acromegaly. However, few data are available on cardiac autonomic functions and sympathovagal balance in acromegalic patients. OBJECTIVE: The aim of this study was to investigate both the time and frequency domain parameters of Heart Rate Variability (HRV), in order to characterize the cardiac autonomic functions in patients affected by acromegaly. This study correlated anthropometric, metabolic, echocardiographic parameters and blood pressure with those relating to HRV, to identify the main factors responsible for the HRV related alterations possibly present. We also aimed to analyze the effects of the treatment with somatostatin analogues (SSAs) on HRV. MATERIALS AND METHODS: This study enrolled 47 acromegalic patients (23 males, age 49.1 ± 13.5 years) and 37 (13 males) age matched (52.3 ± 13.3 years) healthy subjects. All participants underwent 12-lead 24 h ECG Holter recordings and a HRV analysis of the ECG tracings was performed. The parameters obtained from the time domain analysis of HRV included pNN50, SDNN, SDNN index, SDANN and RMSSD. The power spectral analysis of HRV was obtained by summing powers of the LF (low frequency) and the HF (high frequency) band. Sympathovagal balance was estimated by calculating the LF/HF ratio during 24 h and 15 min of clinostatism. The HRV of 28 acromegalic patients was studied before and after SSAs treatment. RESULTS: Acromegalic patients showed significantly lower SDNN and SDANN compared to controls. Diabetic and non-diabetic acromegalic patients showed decreased SDNN and SDANN, when compared to healthy subjects. Diabetic acromegalic patients had a lower LF/HF ratio during 24 h when compared to non-diabetic acromegalic patients. Similar results were obtained analyzing patients affected by acromegaly and impaired glucose tolerance. SDNN and SDANN were lowered by hypertension in the acromegalic population, when compared to controls, and hypertensive acromegalic patients also displayed a decreased LF/HF ratio during 24 h when compared to normotensive acromegalic subjects. Patients with ventricular arrhythmias in Lown classes 3-5 showed a decreased SDANN compared to patients in Lown class 0-2. The treatment with SSAs was able to ameliorate all the time domain parameters of HRV, without altering the 24 h LF/HF ratio. CONCLUSION: Cardiac autonomic functions and sympathovagal balance are altered in patients affected by acromegaly and could be ameliorated by SSAs therapy. HRV analysis allows an estimation of the autonomic sympathovagal balance and may be a useful clinical tool for the cardiac risk stratification in acromegalic patients.

Ambulatory arterial stiffness indices and non-alcoholic fatty liver disease in essential hypertension.

BACKGROUND AND AIM: Non-alcoholic fatty liver disease (NAFLD) has been found to be strongly related to an increased arterial stiffness in patients with essential hypertension, suggesting a pathophysiologic link between major cardiovascular and metabolic abnormalities associated with liver steatosis and the functional and structural alterations of the arterial wall. The aim of our study was to investigate, in a group of essential hypertensive patients without additional cardiovascular risk factors, the relationship between NAFLD and arterial stiffness. METHODS AND RESULTS: Sixty-eight consecutive patients with essential hypertension underwent 24-h ambulatory blood pressure monitoring (ABPM) and were separated according to the presence (n = 40) or absence (n = 28) of NAFLD at liver ultrasonography. The Ambulatory Arterial Stiffness Index (AASI) and Symmetric AASI (Sym-AASI) were derived from ABPM tracings. Patients with diabetes, obesity, hyperlipidaemia or other risk factors for cardiovascular or liver disease were excluded. Hypertensive patients were compared with a normotensive control group.The two hypertensive groups had comparable age, sex distribution and clinic/ABPM blood pressure levels. In hypertensive patients with NAFLD, body mass index, fasting glucose, insulin, homeostasis model of assessment of insulin resistance index and triglyceride levels were higher, whereas plasma adiponectin was lower than in patients without NAFLD. In hypertensive patients, AASI and Sym-AASI were higher (P < 0.001) than in normotensive subjects, but both indices of vascular stiffness were comparable in patients with and without NAFLD. CONCLUSIONS: In essential hypertensive patients without additional cardiovascular risk factors, NAFLD is associated with insulin resistance but not with increased arterial stiffness.

Low serum 25-hydroxyvitamin D levels are associated with left ventricular hypertrophy in essential hypertension.

BACKGROUND AND AIM: Low serum 25-hydroxyvitamin D [25(OH)D] levels may have an important role in predisposing to hypertension and myocardial disease. We investigated the relationship between 25(OH)D and left ventricular (LV) structure and function, assessed by echocardiography, in a series of patients with essential hypertension (EH). METHODS AND RESULTS: Sixty-two newly diagnosed never-treated patients (32 male/30 female), aged 18-65 years, with grade 1-2 hypertension, no diabetes, no obesity, no hyperlipidemia, and no cardiopulmonary, renal, or hepatic disease, were studied. Twenty-four healthy normotensive sex-, age-, BMI-matched subjects served as controls. Hypertensive patients with 25(OH)D deficiency, defined as serum 25(OH)D levels <50 nmol/L, had higher prevalence of LV hypertrophy (LVH) than their 25(OH)D-sufficient counterparts (57.1 vs 17.6%, P = 0.02); no differences between the two groups were found in blood pressure levels as well as in other biochemical and hormone parameters. There was an inverse correlation between LV mass index and 25(OH)D levels (r = -0.366, P < 0.003) and a direct correlation between LV mass index and BMI (r = 0.333, P < 0.006) in the entire hypertensive population. The two variables remained independently associated with LVH at multivariable logistic regression analysis (OR 1.05, P < 0.005 and OR 1.25, P = 0.03, respectively). Prevalence of 25(OH)D deficiency was similar in EH patients and controls (45.1 vs 41.6%, P = 0.89), whereas no correlation between echocardiographic parameters and hormone levels was found. CONCLUSIONS: In the absence of major cardiovascular risk factors, 25(OH)D deficiency is a frequent finding in EH patients and is independently associated with LVH.

Primary aldosteronism and metabolic syndrome.

Hypertension is frequently associated with interrelated risk factors of metabolic origin, including abdominal obesity, dyslipidemia, and alterations in glucose homeostasis, all promoting the pathogenesis of arteriosclerosis. Clustering of these risk factors, defined as metabolic syndrome, is associated with an overall high cardiovascular risk profile. This article reviews current knowledge regarding the prevalence and characteristics of the metabolic syndrome in primary aldosteronism, and discusses a possible pathophysiological link between aldosterone and its individual components other than hypertension. An abnormal glucose metabolism due to insulin resistance appears to be linked to aldosterone overproduction, and seems the major contributor to metabolic dysfunction in primary aldosteronism. Impairment of insulin action may be also due to concurrent environmental factors (hypokalemia?), and/or it might occur in compartments other than fat tissue (liver? skeletal muscle?). Higher rates of cardiovascular events reported in primary aldosteronism could be due in part to the increased prevalence of the metabolic syndrome in this disorder. Regression of glucometabolic complications after the cure of aldosterone excess should be confirmed by larger studies, and the influence on the natural history of primary aldosteronism by using agents potentially able to correct metabolic abnormalities should be further explored.

Aldosterone does not modify gene expression in human endothelial cells.

The toxic effects of aldosterone on the vasculature, and in particular on the endothelial layer, have been proposed as having an important role in the cardiovascular pathology observed in mineralocorticoid-excess states. In order to characterize the genomic molecular mechanisms driving the aldosterone-induced endothelial dysfunction, we performed an expression microarray on transcripts obtained from both human umbilical vein endothelial cells and human coronary artery endothelial cells stimulated with 10 - 7 M aldosterone for 18 h. The results were then subjected to qRT-PCR confirmation, also including a group of genes known to be involved in the control of the endothelial function or previously described as regulated by aldosterone. The state of activation of the mineralocorticoid receptor was investigated by means of a luciferase-reporter assay using a plasmid encoding a mineralocorticoid and glucocorticoid-sensitive promoter. Aldosterone did not determine any significant change in gene expression in either cell type both in the microarray and in the qRT-PCR analysis. The luciferase-reporter assay showed no activation of the mineralocorticoid receptor following aldosterone stimulation. The status of nonfunctionality of the mineralocorticoid receptor expressed in cultured human umbilical and coronary artery endothelial cells does not allow aldosterone to modify gene expression and provides evidence against either a beneficial or harmful genomic effect of aldosterone on healthy endothelial cells.

Insulin signaling in adipose tissue of patients with primary aldosteronism.

OBJECTIVE: We studied phosphorylation of insulin-receptors substrate downstream molecules: 1) in the ex-vivo visceral adipose tissue (VAT) of patients with aldosterone-producing adenoma (APA) (no.=7) and non-functioning adenoma (NFA) (no.=7) undergoing laparoscopic adrenalectomy; 2) in aldosterone-treated sc adipocytes of subjects (no.=5) who requested abdominoplasty. PATIENTS AND METHODS: Western blotting was used to detect phosphorylation of Akt and extracellular signal-regulated kinase (ERK) 1/2 in VAT from APA and NFA patients, and in subcutaneous adipocytes pre-treated with different aldosterone concentrations. Phosphorylation of Akt and ERK1/2 was similar in VAT of patients with APA and NFA. Pre-treatment in adipocytes with both physiological (1 nM) and pharmacological (10 µM) doses of aldosterone did not affect basal or insulin-induced phosphorylation of Akt and ERK1/2. CONCLUSIONS: Our data give further evidence that insulin signaling in human VAT is not affected by primary aldosterone overproduction.

Progress in primary aldosteronism: present challenges and perspectives.

Primary Aldosteronism (PA) is a disorder of the adrenal zona glomerulosa (ZG) in which aldosterone secretion is increased and is relatively autonomous of normal regulatory mechanisms. A recent conference in Munich organized by Prof. Reincke addressed advances and challenges related to the screening, diagnosis, and identification of uni- and bilateral involvement of the diseased adrenal of PA. Some infrequently addressed issues are described herein. We postulate that most cases of PA are due to the activation by unknown mechanisms of subset of cells resulting in the formation of a multiple foci or nodules of hyperactive zona glomerulosa cells. This implies that one or several yet unidentified stimuli can drive aldosterone overproduction, as well as the proliferation of aldosterone-producing cells. Current diagnostic procedures allow to determine whether inappropriate aldosterone production is driven by one or both adrenal glands and thus to establish optimal treatment.

Cardiovascular autonomic function in Cushing's syndrome.

Cardiac autonomic dysfunction is associated with increased cardiovascular mortality. No data on sympathovagal balance are available in patients with Cushing's syndrome, in whom cardiovascular risk is high. We studied 10 patients with newly diagnosed Cushing's syndrome (1 male/9 females; age mean+/-SD, 47+/-10 yr) and 10 control subjects matched for age, sex, body mass index, and cardiovascular risk factors. In both groups there were 7 patients with arterial hypertension, 3 with diabetes mellitus, and 2 with obesity. Cardiac autonomic function was evaluated by analysis of short time heart rate variability (HRV) measures in frequency domain over 24-h, daytime, and nighttime. The 24-h ambulatory blood pressure monitoring and echocardiography were also performed. In comparison with controls, patients with Cushing's syndrome had lower 24-h (1.3+/-0.6 vs 3.7+/-1.5, mean+/-SD, p<0.01), daytime (2.0+/-1.4 vs 4.5+/-1.6, p<0.01), and night-time (1.0+/-0.4 vs 3.5+/-2.3, p<0.01) low-frequency/ high frequency (LF/HF) power ratio. In the presence of similar LF power, the difference was due to elevation in HF power in Cushing's syndrome compared to controls: 24-h, 12.7+/-6.7 vs 5.8+/-2.8, p<0.01; daytime, 10.2+/-7.3 vs 4.5+/-2.1, p<0.05; nighttime, 14.2+/-7.0 vs 7.8+/-4.7, p<0.05. Eight Cushing patients vs 4 controls had a non-dipping blood pressure profile. At echocardiography, Cushing patients had a greater left ventricular mass index and/or relative wall thickness, and impaired diastolic function, compared with controls. Compared to controls, patients with Cushing's syndrome showed a sympathovagal imbalance, characterized by a relatively increased parasympathetic activity. Whether this autonomic alteration is meant to counterbalance cortisol-induced effects on blood pressure and cardiac structure/function or has a different pathophysiological significance is still unknown.

Non-alcoholic fatty liver disease is associated with left ventricular diastolic dysfunction in essential hypertension.

BACKGROUND AND AIM: Insulin resistance is recognized as the pathophysiological hallmark of non-alcoholic fatty liver disease (NAFLD). A relation between insulin sensitivity and left ventricular morphology and function has been reported in essential hypertension, where a high prevalence of NAFLD has been recently found. We investigated the inter-relationship between left ventricular morphology/function, metabolic parameters and NAFLD in 86 never-treated essential hypertensive patients subdivided in two subgroups according to the presence (n = 48) or absence (n = 38) of NAFLD at ultrasonography. METHODS AND RESULTS: The two groups were similar as to sex, age and blood pressure levels. No patient had diabetes mellitus, obesity, hyperlipidemia, or other risk factors for liver disease. Body mass index, waist circumference, triglycerides, glucose, insulin, homeostasis model of assessment index for insulin resistance (HOMA-IR), aspartate aminotransferase and alanine aminotransferase were higher and adiponectin levels were lower in patients with NAFLD than in patients without NAFLD, and were associated with NAFLD at univariate analysis. Patients with NAFLD had similar prevalence of left ventricular hypertrophy compared to patients without NAFLD, but a higher prevalence of diastolic dysfunction (62.5 vs 21.1%, P < 0.001), as defined by E/A ratio <1 and E-wave deceleration time >220 ms. Diastolic dysfunction (P = 0.040) and HOMA-IR (P = 0.012) remained independently associated with NAFLD at backward multivariate analysis. CONCLUSIONS: Non-alcoholic fatty liver disease was associated with insulin resistance and abnormalities of left ventricular diastolic function in a cohort of patients with essential hypertension, suggesting a concomitant increase of metabolic and cardiac risk in this condition.

Conceptual basis and methodology of the SOPHIA study.

To clarify the role of gene polymorphisms on the effect of losartan and losartan plus hydrochlorothiazide on blood pressure (primary end point) and on cardiac, vascular and metabolic phenotypes (secondary end point) after 4, 8, 12, 16 and 48 weeks treatment, an Italian collaborative study - The Study of the Pharmacogenomics in Italian hypertensive patients treated with the Angiotensin receptor blocker losartan (SOPHIA) - on never-treated essential hypertensives (n = 800) was planned. After an 8 week run-in, losartan 50 mg once daily will be given and doubled to 100 mg at week +4 if blood pressure is more than 140/90 mmHg. Hydroclorothiazide 25 mg once daily at week +8 and amlodipine 5 mg at week +16 will be added if blood pressure is more than 140/90 mmHg. Cardiac mass (echocardiography), carotid intima-media thickness, 24 h ambulatory blood pressure, homeostatic model assessment (HOMA) index, microalbuminuria, plasma renin activity and aldosterone, endogenous lithium clearance, brain natriuretic peptide and losartan metabolites will be evaluated. Genes of the renin-angiotensin-aldosterone system, salt sensitivity, the beta-adrenergic system and losartan metabolism will be studied (Illumina custom arrays). A whole-genome scan will also be performed in half of the study cohort (1M array, Illumina 500 GX beadstation).

The role of 21-hydroxylase in the pathogenesis of adrenal masses: review of the literature and focus on our own experience.

An exaggerated response of 17- hydroxyprogesterone (17-OHP) to exogenous ACTH stimulation has been found in 30 to 70% of patients with incidentally discovered adrenal tumors, supporting the concept that congenital 21- hydroxylase deficiency may be a predisposing factor for adrenocortical tumorigenesis. Decreased expression of 21-hydroxylase gene has been observed in sporadic non-functioning adrenocortical adenomas and adrenocortical carcinomas, in agreement with the reduced steroidogenic activity found in these types of tumors. Screening studies for the presence of mutations in CYP21A2 gene, encoding 21-hydroxylase, in patients with sporadic adrenocortical tumors yielded discordant results. Overall, a higher frequency of germline 21-hydroxylase mutation carriers has been found among patients with adrenal tumors, including incidentalomas, than in the general population. However, the presence of mutations did not correlate with endocrine test results and tumor mass features, suggesting that 21-hydroxylase deficiency does not represent a relevant mechanism in adrenal tumorigenesis. Mechanisms leading to reduced 21-hydroxylase expression and activity are still unknown.

P450c17 deficiency: clinical and molecular characterization of six patients.

CONTEXT: The characteristics of P450c17 deficiency include 46,XY disorder of sex development, hypertension, hypokalemia, and lack of pubertal development. OBJECTIVE: To better understand this rare enzymatic deficiency, we analyzed the CYP17A1 gene in six affected patients. DESIGN AND PATIENTS: We examined six patients, five 46,XY, and one 46,XX (age 9-29 yr) with complete lack of masculinization (female infantile external genitalia, no uterus) and delayed puberty, respectively, and different degrees of hypertension. MAIN OUTCOME MEASUREMENTS: Genotype-phenotype correlation was measured. RESULTS: Four homozygote mutations were identified by direct sequencing of the CYP17A1 gene corresponding to an alanin 302-proline (A302P) exchange; the loss of lysine 327 (K327del); the deletion of glutamate 331 (E331del); and the replacement of arginine 416 with a histidine (R416H). Both P450c17 activities were abolished in all the mutant proteins, except one, when expressed in COS1 cells. The E331del-mutated P450c17 retained 17alpha-hydroxylase activity. The mutant proteins were normally expressed, suggesting that the loss of enzymatic activity is not due to defects of synthesis, stability, or localization of P450c17 proteins. CONCLUSION: These studies confirm lack of masculinization in 46,XY individuals as the pathognomic sign of the complete P450c17 deficiency. In XX individuals P450c17 deficiency should be considered in cases of delayed puberty. Age of onset and the severity of hypertension do not seem to be constant. Careful examination of long-term follow-ups in two of our patients suggested to us that estrogen treatment in P450c17-deficient patients might worsen the enzymatic defect, leading to aggravation of the hypertension.