Multicenter retro-prospective observational study on chronic hypoparathyroidism and rhPTH (1-84) treatment.

PURPOSE: The main purpose of this study was to investigate the effects of 12 months of rhPTH (1-84) (Natpar®) treatment in a cohort of patients selected according to the indications of hypoparathyroidism guidelines. The use of recombinant human PTH (1-84) [rhPTH (1-84)] is approved as hormonal replacement therapy in patients with hypoparathyroidism not adequately controlled with conventional therapy. METHODS: It is a multicenter, observational, retro-prospective, open label study. Eleven Italian Endocrinological centers, members of Hypoparathyroidism Working Group of the Italian Society of Endocrinology (HypoparaNET) were involved. Main outcome measures were serum and urinary calcium and phosphate concentration, calcium-phosphate product, renal function, oral calcium and vitamin D doses, and clinical manifestations. RESULTS: Fourteen adult subjects, affected by chronic hypoparathyroidism, were treated with rhPTH (1-84) for 12 months. At 12 months of rhPTH (1-84) treatment, 61.5% of patients discontinued calcium supplement and 69.2% calcitriol. Mean albumin-adjusted total serum calcium levels quickly normalized after initiation of rhPTH (1-84) treatment compared to baseline (p = 0.009), remaining in the normal range until 12 months. Rare hypo-hypercalcemia episodes were reported. Renal function was maintained normal and no renal complications were reported. Serum and urinary phosphate and urinary calcium were maintained in the normal range. Mean phosphatemia levels linearly decreased from 3 months up to 12 months compared to baseline (p = 0.014). No severe adverse events were described. CONCLUSIONS: Biochemical and clinical results confirm the efficacy and safety of rhPTH (1-84) therapy, which represents an important option for hypoparathyroid patients unresponsive to conventional therapy.

POR polymorphisms are associated with 21 hydroxylase deficiency.

PURPOSE: Genotype-phenotype correlation in congenital 21 hydroxylase deficiency is strong but by no means absolute. Indeed, clinical and hormonal features may vary among patients carrying similar CYP21A2 mutations, suggesting that modifier genes may contribute to the phenotype. Aim of the present study was to evaluate whether polymorphisms in the p450  oxidoreductase (POR) gene may affect clinical features in patients with 21 hydroxylase deficiency METHODS: Sequencing of the POR gene was performed in 96 patients with 21 hydroxylase deficiency (49 classic, 47 non-classic) and 43 control subjects. RESULTS: Prevalence of POR polymorphisms in patients with 21 hydroxylase was comparable to controls and known databases. The rs2228104 polymorphism was more frequently associated with non-classic vs classic 21 hydroxylase deficiency (allelic risk 7.09; 95% C.I. 1.4-29.5, p < 0.05). Classic 21 hydroxylase-deficient carriers of the minor allele in the rs2286822/rs2286823 haplotype presented more frequently the salt-wasting form (allelic risk 1.375; 95% C.I. 1.138-1.137), more severe Prader stage at birth (allelic risk 3.85; 95% C.I. 3.78-3.92), higher ACTH levels, and younger age at diagnosis. CONCLUSIONS: Polymorphisms in the POR gene are associated with clinical features of 21 hydroxylase deficiency both as regards predisposition to classic vs non-classic forms and severity of classic adrenal hyperplasia.

Functional and structural analysis of four novel mutations of CYP21A2 gene in Italian patients with 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder mainly caused by defects in the 21-hydroxylase gene (CYP21A2), coding for the enzyme 21-hydroxylase (21-OH). About 95% of the mutations arise from gene conversion between CYP21A2 and the inactive pseudogene CYP21A1P: only 5% are novel CYP21A2 mutations, in which functional analysis of mutant enzymes has been helpful to correlate genotype-phenotype. In the present study, we describe 3 novel point mutations (p.L122P, p.Q481X, and p.E161X) in 3 Italian patients with CAH: the fourth mutation (p.M150R) was found in the carrier state. Molecular modeling suggests a major impact on 21-hydroxylase activity, and functional analysis after expression in COS-7 cells confirms reduced enzymatic activity of the mutant enzymes. Only the p.M150R mutation affected the activity to a minor extent, associated with NC CAH. CYP21A2 genotyping and functional characterization of each disease-causing mutation has relevance both for treatment and genetic counseling to the patients.

Tryptophan hydroxylase autoantibodies as markers of a distinct autoimmune gastrointestinal component of autoimmune polyendocrine syndrome type 1.

BACKGROUND: Autoantibodies to tryptophan hydroxylase (TPHAbs) directed against serotonin-producing enterochromaffin cells (EC) have been reported in autoimmune-polyendocrine-syndrome type 1 (APS-1) patients with gastrointestinal dysfunction (GID). Serotonin plays a critical role in enteric function and its peripheral blood levels reflect serotonin release from the gastrointestinal tract. AIMS: We test the hypothesis that TPHAbs mark a distinct autoimmune component of APS-1 characterized by an autoimmune attack toward EC, which results in clinical GID. METHODS: TPHAbs were measured in 64 APS-1 patients. Endoscopy with gastric (antrum/body) and duodenal biopsy was carried in 16 TPHAbs+ patients (8 with and 8 without GID) and in 2 TPHAbs- patients (without GID). Immunohistochemistry of biopsy specimens was carried out using antibodies to serotonin, chromogranin-A, CD3, CD4, CD8, and CD20. Serotonin serum levels were measured in TPHAbs+ and TPHAbs- patients who had endoscopy. RESULTS: Thirty-seven of 64 patients were TPHAbs+ (11/12 with GID and 26/52 without GID; P < .001). Gastric and duodenal biopsies in all 8 TPHAb+ patients with GID showed lymphocytic infiltration with increased CD3+CD8+ intraepithelial lymphocytes and absence of EC. Furthermore, mean serotonin serum levels were below the normal range in TPHAb+ patients with GID (P < .01). In 8 TPHAb+ patients without GID gastric and duodenal biopsies showed different grades of inflammatory infiltration and reduced number of EC. Mean serotonin serum levels were near the lower limit of the normal range. In all TPHAbs+ patients the biopsies showed a reduced number of chromogranin-A positive cells consistent with enteroendocrine cells depletion. TPHAbs- patients without GID showed normal gastrointestinal mucosa and serotonin serum levels. CONCLUSIONS: TPHAbs appear to be markers of a distinct autoimmune component of APS-1. Progressive involvement of the gastrointestinal EC leads to the transition from preclinical to clinical disease, characterized by GID and reduced serotonin serum levels.

Autoimmune-polyendocrinopathy-candidiasis-ectodermal-dystrophy in Calabria: clinical, immunological and genetic patterns.

UNLABELLED: Autoimmune polyendocrinopathy-candidiasis-ectodermal- dystrophy (APECED), also known as autoimmune polyendocrine syndrome type 1 (APS-1), is a very rare disease. Diagnosis requires the presence of at least two of three major clinical features: chronic mucocutaneous candidiasis, chronic hypoparathyroidism, and Addison's disease. DESIGN: In this study, we analyzed Autoimmune Regulator (AIRE) gene mutations and genotype-phenotype correlation in APECED patients originating from Calabria, a region in the south of Italy. PATIENTS AND METHODS: Four patients and their first-degree relatives were evaluated for clinical manifestations, autoantibody presence and AIRE gene mutations. RESULTS: Three patients carried a homozygous W78R mutation on exon 2, typical of patients with APECED from Apulia; the fourth patient had a homozygous R203X mutation on exon 5, typical of APECED patients from Sicily. Clinical disease expression showed wide variability. Analysis of relatives allowed the identification of 6 heterozygotes, none of whom showed major findings of APECED. CONCLUSIONS: No AIRE gene mutations specific to Calabria were found in patients with APS-1, but mutations similar to those in patients from Apulia and Sicily. Heterozygosity for AIRE gene mutation is not associated with major findings of APECED.

Evidence for epigenetic abnormalities of the androgen receptor gene in foreskin from children with hypospadias.

CONTEXT: Hypospadias is a malformation of the penis due to an incomplete development of the male urethra, the exact etiology of which in the majority of cases remains unknown. OBJECTIVE: The objective of the study was to assess whether defects of the androgen receptor (AR) gene (CAG repeats and methylation pattern) and DNA methyltransferases (DNMT) family are present in hypospadic patients. DESIGN: CAG repeats length, methylation status, and expression of the AR gene were analyzed. The DNMT family was studied at the protein level and the DNMT3A sequenced. SETTING: The study was performed at a pediatric endocrinology referral clinic. PATIENTS OR OTHER PARTICIPANTS: Twenty boys with isolated glandular hypospadias and 20 age-matched control children undergoing a surgical procedure for circumcision were studied. MAIN OUTCOME MEASURE(S): CAG repeats length and AR methylation pattern in PBLs and foreskin tissue, DNMT expression and sequencing in patients and controls, and in vitro studies in cultured fibroblasts were measured. RESULTS: AR gene methylation in foreskin tissues from patients with hypospadias was higher than in normal children. AR expression in foreskin tissue of hypospadic patients was lower than in controls, whereas the DNMT3A protein level was significantly higher in patients than controls. In cultured fibroblasts, both dihydrotestosterone and testosterone significantly reduced AR gene methylation and DNMT3A expression in a dose-dependent fashion and increased AR expression. CONCLUSION: The AR gene in target tissues from patients with hypospadias is more methylated than in control children, resulting in a decreased expression of the AR. The mechanism underlying the modulation of the AR gene expression seems to be mediated by DNMT3A. This epigenetic alteration of the AR gene might be involved in the pathogenesis of hypospadias.

Long-term auxological and pubertal outcome of patients with hereditary insulin-like growth factor-I deficiency (Laron and growth hormone-gene deletion syndrome) treated with recombinant human insulin-like growth factor-I.

BACKGROUND: GH-IGF-I axis is mainly involved in the complex process of somatic growth but emerging evidence suggests that it also influences hypothalamic-pituitary-gonadal (HPG) function. SUBJECTS: We report some data regarding long-term auxological and pubertal outcome of five female patients with hereditary forms of GH-IGF-I deficiency (Laron and GH-gene deletion syndrome) and a mean age of 23.4±5.3 yr (range 19-32). METHODS: All the patients received recombinant human IGF-I (rhIGF-I, Pharmacia and Upjohn, Stockholm, Sweden, and rhIGF-I, Genentech, San Francisco, CA, USA) from a mean age of 8.6 yr (range 3.2-14.2) up to the final height. RESULTS: Final height was very disappointing (≤ -5.0 SD scores) and lower than target height in all the patients. Pubertal onset was delayed in most of them but menarche occurred spontaneously in all the patients. Median age at menarche was 15.1 yr. Menstrual cycles were regular for several years. Median duration of gynecological follow- up was 8.3 yr with the longest span of 17.2 yr. CONCLUSION: We can assert that GH-IGF-I axis has an essential role in promoting linear growth in humans and its physiological action cannot be replaced by pharmacological treatment in most patients with hereditary forms of IGF-I insufficiency as demonstrated by their subnormal final height. Our clinical observations can also support an essential role of IGF-I in genitalia growth but not in the function of HPG axis as demonstrated by the maintenance of regular menstrual cycles in the presence of subnormal levels of IGF-I after treatment discontinuation.

GH secretion in a cohort of children with pseudohypoparathyroidism type Ia.

Pseudohypoparathyroidism type Ia (PHP-Ia) is characterized by Albright's hereditary osteodistrophy (AHO) and resistance to hormones that act via the alpha subunit of the Gs protein (Gsalpha) protein, ie PTH, TSH, FSH/LH, and, as recently described in limited series, GHRH. However, the current lack of data on GHRH secretion, obesity and short stature included in the AHO phenotype hampers interpretation of GH secretory status and its effects on these subjects. We evaluated GH secretion after GHRH plus arginine (Arg) stimulus, IGF-I levels and anthropometric features in an exclusively pediatric population of 10 PHP-Ia subjects. Of our PHP-Ia children, 5 out of 10 (50%) showed impaired GH responsiveness to the provocative test, with a lower prevalence than the 75-100% previously reported. A negative correlation (p=0.024) was found between GH secretion and body mass index (BMI), whereas no correlation emerged between GH and IGF-I values (p=0.948). Height and growth velocity did not significantly differ between GH-deficient and GH-sufficient subjects. In the 5 GH-deficient patients, GHRH resistance could arguably be responsible for hormonal impairment; however, 3 of them were obese, showing normal stature and IGF-I levels: the increased BMI in these subjects could influence GH secretion and its effects. In conclusion, GH deficiency is frequent among PHP-Ia children and its prevalence is variable, two factors indicating that GH secretory testing should be part of the routine management of this patient group. It could be argued that GHRH resistance is the pathogenetic mechanism in most patients, but further studies on GHRH secretion are needed to define which values can be considered as raised. Lastly, because BMI has been indicated as a major determinant of evoked adult GH response to provocative testing, GH levels related to increased BMI also in childhood could be helpful in defining GH assessment in obese or overweight PHP-Ia children.

Aromatase is differentially expressed in peripheral blood leukocytes from children, and adult female and male subjects.

OBJECTIVE: Aromatase, the key enzyme involved in estrogen synthesis, is expressed in a variety of cells and tissues including human peripheral blood leukocytes (PBLs). The present study was designed to evaluate PBL aromatase gene expression in male and female subjects of different age groups. In addition, differences in gene expression during the follicular and luteal phase of the menstrual cycle in women, and before and after testosterone administration in men, were estimated. DESIGN: Aromatase mRNA and protein were measured in PBLs obtained from young (n = 10) and postmenopausal women (n = 10), men (n = 15), and prepubertal children (n = 10). Aromatase mRNA and protein were also measured during the follicular and luteal phases of the menstrual cycle in women, and before and after the intramuscular administration of 250 mg testosterone enanthate in men. METHODS AND RESULTS: Aromatase mRNA measured by real-time PCR in PBLs from women during the follicular phase was significantly higher than during the luteal phase of the menstrual cycle (P < 0.05). In men, PBL aromatase mRNA values increased significantly following testosterone administration (P < 0.05). PBL mRNA aromatase levels in women during the follicular phase and men after testosterone administration were significantly higher (one-way ANOVA; P < 0.05) than in any other group. Children, postmenopausal women, and women during the luteal phase showed the lowest aromatase mRNA expression. The results of the immunoblot analysis confirmed the data obtained by real-time PCR. A positive correlation between PBL aromatase mRNA values and plasma estradiol and estrone levels during the follicular phase of the menstrual cycle was observed in the group of adult women. No other correlations were found. CONCLUSIONS: The aromatase gene is differentially expressed in PBLs from women, men, and prepubertal children, indicating a sexual dimorphism in the enzyme expression and an important role of sex steroids in the modulation of aromatase gene expression.

Decreased androgen receptor gene methylation in premature pubarche: a novel pathogenetic mechanism?

CONTEXT: Several studies found links between DNA methylation and gene expression. In patients with idiopathic hirsutism, a preferential methylation of the of shorter androgen receptor (AR) alleles was hypothesized to be responsible for the abnormal hair growth. OBJECTIVE: The objective of this study was to assess whether abnormalities in the AR function in both peripheral blood leukocytes (PBLs) and androgen target tissues are present in children with premature pubarche (PP). DESIGN: Human DNA was extracted from PBLs and pubic hair and CAG repeats length and methylation status of the AR gene were analyzed. SETTING: The study was performed at a Pediatric Endocrinology referral clinic. PATIENTS: Twenty-five girls with PP, 23 prepubertal children, and 10 girls with Tanner stage II pubertal development were studied. MAIN OUTCOME MEASURE: The main outcome measures were CAG repeat length and AR methylation pattern in PBLs and pubic hair. RESULTS: In PBLs from PP patients, AR gene methylation was significantly lower (P < 0.01) than that of prepubertal children and similar to that of girls with Tanner II stage pubertal development. A negative correlation between AR gene methylation in PBLs and the age of normal children was detected. PATIENTS with PP exhibited a hair follicle AR methylation pattern similar to that of Tanner stage II girls. The mean number of CAG repeats was lower in PP patients than in prepubertal and Tanner stage II girls, although it was within the normal range for the general population in both groups. CONCLUSIONS: The increased AR gene activity observed in PP patients, as indicated by the reduced AR gene methylation pattern, together with the presence of shorter CAG repeats, might lead to hypersensitivity of the hair follicles to steroid hormones and therefore to the premature development of pubic hair.

Evaluation of the circadian profile of peripheral plasma galanin concentrations in normal subjects.

Galanin administration can influence pituitary function principally resulting in an increase in GH secretion. However, the role of circulating GAL levels in human endocrine function is still unknown. In the present study we simultaneously measured the circadian profiles of GAL, ACTH and GH in peripheral blood of ten adult subjects. Plasma samples were collected through an intravenous catheter at 0800, 1200, 1600, 2000, 2200, 2400, 0200, 0400 hours. The results were statistically evaluated by the cosinor analysis technique. A significant circadian rhythm of both plasma ACTH (p < 0.001) and GH levels (p < 0.03) was found with acrophases occurring at 0753 hrs and 0131 hrs for ACTH and GH, respectively. On the contrary, no significant rhythm was found in plasma GAL levels, indicating that no correlations exist between GAL and either GH or ACTH circadian profiles. Furthermore, the simultaneous assay of both GAL and GH plasma levels during a nocturnal frequent sampling performed in four volunteers showed the presence of peaks in GAL levels which, however, were not concomitant to the peaks in GH levels. These data demonstrate the lack of rhythmicity in the circadian profile of plasma GAL levels in healthy human subjects. The role of GAL in human endocrine function remains unknown and these results suggest that, in spite of the well documented increase in plasma GH concentrations following the intravenous administration of GAL, physiologically circulating levels of GAL are likely not involved in the regulation of GH secretion.

Leptin, cortisol, and GH secretion interactions in short normal prepubertal children.

The hormonal regulation of the ob gene and leptin secretion in humans is still unclear. To investigate the interactions among leptin, cortisol, and GH, we analyzed and time-cross-correlated their spontaneous 24-h secretion in 12 short normal prepubertal children of both sexes (6 females and 6 males). Time-cross-correlation analyses demonstrated that leptin and cortisol were correlated in both a negative and positive fashion. The negative correlation, with cortisol leading leptin by 4 and 3 h for boys and girls, respectively, might reflect the stimulatory effect of CRH on the sympathetic system, which inhibits leptin secretion; the positive correlation, with leptin leading cortisol by 6 and 5 h for boys and girls, respectively, might reflect a direct effect of leptin on CRH secretion in the hypophyseal portal system. Time-cross-correlation analyses also showed a strong positive correlation between GH and leptin concentrations, with GH leading leptin by 5 and 2 h for boys and girls, respectively, suggesting a possible direct leptin-releasing effect of GH on adipocytes. We conclude that cross-correlation analyses of 24-h hormone secretions under baseline physiological conditions suggest that the hypothalamic-pituitary-adrenal axis might have a prevailing inhibitory effect on leptin secretion, whereas leptin might exert a positive effect on the hypothalamic-pituitary-adrenal axis. The relation between GH and leptin could be a direct one and characterized prevalently by a positive effect of GH on leptin secretion. Further investigations using different experimental systems are needed to ascertain the validity of these mathematically educed conclusions.

Leptin inhibits steroid biosynthesis by human granulosa-lutein cells.

Absence of leptin secretion compromises reproductive function and fertility in the ob/ob mouse which, when given leptin, shows a rise in serum LH levels and becomes fertile. Recently, the long and active isoform of the leptin receptor was detected in the ovary, indicating that leptin may also show direct gonad-related activity. To examine this, we studied the effect of graded doses of human leptin on estradiol (E2) and progesterone (P4) concentrations in the culture media of human granulosa-lutein cells obtained from follicular fluid of women undergoing in vitro fertilization. We also evaluated the mRNA expression of steroidogenic acute regulatory protein (StAR), aromatase, and cytochrome P450 17alpha (CYP17) in these cells at baseline and after exposure to leptin. Estradiol levels were significantly decreased in the media 24 hours after incubation of the cells with increasing hLeptin concentrations (10(-11) - 10(-7) mol/l). The maximal 30% decrease in E2 production was caused by the 10(-9) mol/l hLeptin concentration; however, P4 levels in the media were not influenced by leptin. Exposure of granulosa-lutein cells to 10(-9) mol/l hLeptin did not produce any measurable changes on StAR, aromatase, or CYP17 mRNA expression. When hLeptin (10(-9) mol/l) was co-incubated with increasing concentrations of hCG (1.25 - 10 mlU/ml), IGF-II (15-60 ng/ml) or 1-6 desaminated IGF-II (deslGF-II; 15-60 ng/ml), it did not modify the elevation of E2 concentrations caused by each of the different stimuli. We conclude that leptin suppresses E2 secretion by human granulosa-lutein cells but does not impair the stimulatory effects of hCG and IGFs on these cells. Leptin may play a minor, but direct regulatory role on unstimulated human ovarian steroidogenesis by interfering with either the translational or post-translational steps of the baseline CYP17 and/or aromatase synthesis and/or the activation of the enzymes.

An unusual MR presentation of the neurohypophyseal "bright spot" in pituitary dwarfism.

Abnormalities of the hypothalamo-hypophyseal axis are frequently associated with pituitary deficiency. We describe a previously unknown morphological presentation of the ectopic posterior neurohypophyseal "bright spot."

Interactions of leptin and thyrotropin 24-hour secretory profiles in short normal children.

Thyroid hormones and leptin have effects on similar aspects of body homeostasis, such as energy expenditure, thermogenesis, and metabolic efficiency. Thus, the cross-talk between the thyrostat and the lipostat might play a crucial role in the maintenance of body homeostasis. To investigate the relationship between the hypothalamic-pituitary-thyroid (HPT) axis and leptin under physiological conditions, we evaluated the pulsatility and circadian rhythmicity and time-cross-correlated the 24-h secretory patterns of leptin and TSH in 12 short normal prepubertal children (6 girls and 6 boys). In both male and female subjects, leptin was secreted in a pulsatile and circadian fashion, with a nocturnal leptin surge that was more pronounced in males than in females. Mean 24-h leptin levels and total area under the curve were significantly higher in girls than in boys. This was mainly due to the nighttime mean leptin levels and total area under the curve, which were higher than those in boys. The cross-correlated 24-h leptin and TSH levels revealed significant positive and negative correlations. The positive one, of leptin over TSH, suggests a positive feedback regulation by leptin on the HPT axis, which might play an important role in triggering the neuroendocrine response to starvation, including decreased thyroid hormone levels. The negative correlation, of TSH over leptin, could explain the compensatory changes in adipocyte metabolism, and indirectly in circulating leptin levels, in response to alterations in thyroid status. In conclusion, we suggest that under baseline physiological conditions, the HPT axis has a prevailing inhibitory effect on leptin secretion, whereas leptin has a prevailing positive effect on the HPT axis. The sexual dimorphism in leptin levels does not seem to influence in a major way the interactions between the HPT axis and leptin.

Final height in isolated GH deficiency type 1A: effects of 5-year treatment with IGF-I.

Data concerning final height (FH) in isolated growth hormone deficiency type 1A (IGHD1A) are scanty and controversial. In this paper we report the FH outcome of two girls with IGHD1A who were treated either with GH only (first patient) or with GH during the first 8 years and successively with IGF-I (second patient). In the first patient, FH was only slightly subnormal and slightly taller with respect to target height (TH). Surprisingly, FH was severely subnormal and very far from TH in the patient who underwent IGF-I therapy for >5 years: an auxological outcome similar to the one recently reported in the only two cases in the literature of patients with IGHD1A who have been treated with IGF-I until near FH achievement. We conclude that IGHD1A could have a very heterogeneous phenotypic expression in terms of FH and that IGF-I therapy, even though initiated some years before puberty onset and prolonged for more than 5 years, may not be able to ensure the normalization of height prognosis and the achievement of an FH close to TH.