Dehydroepiandrosterone sulfate in the assessment of the hypothalamic-pituitary-adrenal axis

The role of dehydroepiandrosterone-sulfate (DHEA-S) in assessing the integrity of the hypothalamic-pituitary-adrenal (HPA) axis in patients with suspected insufficiency is uncertain.

Germline NF1 mutational spectra and loss-of-heterozygosity analyses in patients with pheochromocytoma and neurofibromatosis type 1

BACKGROUND: Neurofibromatosis type 1 (NF1) is a pheochromocytoma-associated syndrome. Because of the low prevalence of pheochromocytoma in NF1, we ascertained subjects by pheochromocytoma that also had NF1 in the hope of describing the germline NF1 mutational spectra of NF1-related pheochromocytoma. MATERIALS AND METHODS: An international registry for NF1-pheochromocytomas was established. Mutation scanning was performed using denaturing HPLC for intragenic variation and quantitative PCR for large deletions. Loss-of-heterozygosity analysis using markers in and around NF1 was performed. RESULTS: There were 37 eligible subjects (ages 14-70 yr). Of 21 patients with corresponding tumor available, 67% showed somatic loss of the nonmutated allele at the NF1 locus vs. 0 of 12 sporadic tumors (P = 0.0002). Overall, 86% of the 37 patients had exonic or splice site mutations, 14% large deletions or duplications; 79% of the mutations are novel. The cysteine-serine rich domain (CSR) was affected in 35% but the RAS GTPase activating protein domain (RGD) in only 13%. There did not appear to be an association between any clinical features, particularly pheochromocytoma presentation and severity, and NF1 mutation genotype. CONCLUSIONS: The germline NF1 mutational spectra comprise intragenic mutations and deletions in individuals with pheochromocytoma and NF1. NF1 mutations tended to cluster in the CSR over the RAS-GAP domain, suggesting that CSR plays a more prominent role in individuals with NF1-pheochromocytoma than in NF1 individuals without this tumor. Loss-of-heterozygosity of NF1 markers in NF1-related pheochromocytoma was significantly more frequent than in sporadic pheochromocytoma, providing further molecular evidence that pheochromocytoma is a true component of NF1.

Pseudodominant Inheritance of Goitrous Congenital Hypothyroidism Caused by TPO Mutations: Molecular and in Silico Studies

Context and Objective: Most cases of goitrous congenital hypothyroidism (CH) from thyroid dyshormonogenesis 1) follow a recessive mode of inheritance and 2) are due to mutations in the thyroid peroxidase gene (TPO). We report the genetic mechanism underlying the apparently dominant inheritance of goitrous CH in a nonconsanguineous family of French Canadian origin. Design, Setting, and Participants: Two brothers identified by newborn TSH screening had severe hypothyroidism and a goiter with increased (99m)Tc uptake. The mother was euthyroid, but the father and two paternal uncles had also been diagnosed with goitrous CH. After having excluded PAX8 gene mutations, we hypothesized that the underlying defect could be TPO mutations. Results: Both compound heterozygous siblings had inherited a mutant TPO allele carried by their mother (c.1496delC; p.Pro499Argfs2X), and from their father, one brother had inherited a missense mutation (c.1978C-->G; p.Gln660Glu) and the other an insertion (c.1955insT; p.Phe653Valfs15X). The thyroid gland of one uncle who is a compound heterozygote for TPO mutations (p.Phe653Valfs15X/p.Gln660Glu) was removed because of concurrent multiple endocrine neoplasia type 2A. Immunohistochemistry revealed normal TPO staining, implying that Gln660Glu TPO is expressed properly. Modeling of this mutant in silico suggests that its three-dimensional structure is conserved, whereas the electrostatic binding energy between the Gln660Glu TPO and its heme group becomes repulsive. Conclusion: We report a pedigree presenting with pseudodominant goitrous CH due to segregation of three different TPO mutations. Although goitrous CH generally follows a recessive mode of inheritance, the high frequency of TPO mutations carriers may lead to pseudodominant inheritance.

Influence of growth hormone (GH) receptor deletion of exon 3 and full-length isoforms on GH response and final height in patients with severe GH deficiency

CONTEXT: A polymorphism of the GH receptor (GHR) gene resulting in genomic deletion of exon 3 (GHR-d3) has been associated with responsiveness to GH therapy. However, the data reported so far do vary according to the underlying condition, replacement dose, and duration of the treatment. OBJECTIVE, DESIGN: The aim of this study was to analyze the impact of the GHR genotypes in terms of the initial height velocity (HV) resulting from treatment and the impact upon adult height in patients suffering from severe isolated GH deficiency. CONTROLS, PATIENTS, SETTING: A total of 181 subjects (peak stimulated GH

Exon splice enhancer mutation (GH-E32A) causes autosomal dominant growth hormone deficiency

CONTEXT AND OBJECTIVE: Alteration of exon splice enhancers (ESE) may cause autosomal dominant GH deficiency (IGHD II). Disruption analysis of a (GAA) (n) ESE motif within exon 3 by introducing single-base mutations has shown that single nucleotide mutations within ESE1 affect pre-mRNA splicing. DESIGN, SETTING, AND PATIENTS: Confirming the laboratory-derived data, a heterozygous splice enhancer mutation in exon 3 (exon 3 + 2 A-->C) coding for GH-E32A mutation of the GH-1 gene was found in two independent pedigrees, causing familial IGHD II. Because different ESE mutations have a variable impact on splicing of exon 3 of GH and therefore on the expression of the 17.5-kDa GH mutant form, the GH-E32A was studied at the cellular level. INTERVENTIONS AND RESULTS: The splicing of GH-E32A, assessed at the protein level, produced significantly increased amounts of 17.5-kDa GH isoform (55% of total GH protein) when compared with the wt-GH. AtT-20 cells coexpressing both wt-GH and GH-E32A presented a significant reduction in cell proliferation as well as GH production after forskolin stimulation when compared with the cells expressing wt-GH. These results were complemented with confocal microscopy analysis, which revealed a significant reduction of the GH-E32A-derived isoform colocalized with secretory granules, compared with wt-GH. CONCLUSION: GH-E32A mutation found within ESE1 weakens recognition of exon 3 directly, and therefore, an increased production of the exon 3-skipped 17.5-kDa GH isoform in relation to the 22-kDa, wt-GH isoform was found. The GH-E32A mutant altered stimulated GH production as well as cell proliferation, causing IGHD II.

Evaluation of the biological activity of a growth hormone (GH) mutant (R77C) and its impact on GH responsiveness and stature

CONTEXT AND OBJECTIVE: A single missense mutation in the GH-1 gene converting codon 77 from arginine (R) to cysteine (C) yields a mutant GH-R77C peptide, which was described as natural GH antagonist. DESIGN, SETTING, AND PATIENTS: Heterozygosity for GH-R77C/wt-GH was identified in a Syrian family. The index patient, a boy, was referred for assessment of his short stature (-2.5 SD score) and partial GH insensitivity was diagnosed. His mother and grandfather were also carrying the same mutation and showed partial GH insensitivity with modest short stature. INTERVENTIONS AND RESULTS: Functional characterization of the GH-R77C was performed through studies of GH receptor binding and activation of Janus kinase 2/Stat5 pathway. No differences in the binding affinity and bioactivity between wt-GH and GH-R77C were found. Similarly, cell viability and proliferation after expression of both GH peptides in AtT-20 cells were identical. Quantitative confocal microscopy analysis revealed no significant difference in the extent of subcellular colocalization between wt-GH and GH-R77C with endoplasmic reticulum, Golgi, or secretory vesicles. Furthermore studies demonstrated a reduced capability of GH-R77C to induce GHR/GHBP gene transcription rate when compared with wt-GH. CONCLUSION: Reduced GH receptor/GH-binding protein expression might be a possible cause for the partial GH insensitivity with delay in growth and pubertal development found in our patients. In addition, this group of patients deserves further attention because they could represent a distinct clinical entity underlining that an altered GH peptide may also have a direct impact on GHR/GHBP gene expression causing partial GH insensitivity.

Activation of electrical triggers of atrial fibrillation in hyperthyroidism

A shortening of the atrial refractory period has been considered as the main mechanism for the increased risk of atrial fibrillation in hyperthyroidism. However, other important factors may be involved.

Secretion and degradation of glucagon by HIT cells

HIT cells have been widely used to study synthesis and secretion of insulin. It has been assumed that this cell line secretes no other islet hormones. To ascertain whether HIT cells synthesize, secrete, and degrade glucagon, we examined cell extracts for this peptide and compared secretion and degradation of glucagon and insulin during stimulation of the cells by arginine. Glucagon levels in acid extracts of HIT cells were found to be 0.72 +/- 0.15 pmol/mg protein. Both glucagon and insulin were maximally stimulated in a glucagon/insulin molar ratio of 0.029 by arginine concentrations of 25-50 nM, and the concentration of arginine that provided half-maximum responses for both hormones was approximately 3 mM. Diminution of arginine-induced glucagon secretion was caused by somatostatin, a physiological inhibitor of pancreatic islet alpha-cell function. HPLC was used to authenticate the glucagon levels stimulated by arginine for 60 min and measured by RIA. Thirty-six percent of immunoreactive glucagon was found in the fractions representing authentic glucagon, whereas the remaining 64% eluted earlier. Experiments examining the fate of radiolabeled glucagon exposed to HIT cells revealed time-dependent degradation of the radioisotope to earlier eluting forms, which accounted for approximately 50% of the radioactivity by 60 min and was complete by 18 h, indicating that the early peak detected by RIA represented a metabolite of glucagon. Radioisotopic insulin was degraded more slowly with an apparent half-life of approximately 36 h. We conclude that HIT cells are not only able to synthesize, secrete, and degrade insulin, but also much smaller amounts of glucagon.

Active intestinal calcium transport in the absence of transient receptor potential vanilloid type 6 and calbindin-D9k

To study the role of the epithelial calcium channel transient receptor potential vanilloid type 6 (TRPV6) and the calcium-binding protein calbindin-D9k in intestinal calcium absorption, TRPV6 knockout (KO), calbindin-D9k KO, and TRPV6/calbindin-D(9k) double-KO (DKO) mice were generated. TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice have serum calcium levels similar to those of wild-type (WT) mice ( approximately 10 mg Ca2+/dl). In the TRPV6 KO and the DKO mice, however, there is a 1.8-fold increase in serum PTH levels (P < 0.05 compared with WT). Active intestinal calcium transport was measured using the everted gut sac method. Under low dietary calcium conditions there was a 4.1-, 2.9-, and 3.9-fold increase in calcium transport in the duodenum of WT, TRPV6 KO, and calbindin-D9k KO mice, respectively (n = 8-22 per group; P > 0.1, WT vs. calbindin-D9k KO, and P < 0.05, WT vs. TRPV6 KO on the low-calcium diet). Duodenal calcium transport was increased 2.1-fold in the TRPV6/calbindin-D9k DKO mice fed the low-calcium diet (P < 0.05, WT vs. DKO). Active calcium transport was not stimulated by low dietary calcium in the ileum of the WT or KO mice. 1,25-Dihydroxyvitamin D3 administration to vitamin D-deficient null mutant and WT mice also resulted in a significant increase in duodenal calcium transport (1.4- to 2.0-fold, P < 0.05 compared with vitamin D-deficient mice). This study provides evidence for the first time using null mutant mice that significant active intestinal calcium transport occurs in the absence of TRPV6 and calbindin-D9k, thus challenging the dogma that TRPV6 and calbindin-D9k are essential for vitamin D-induced active intestinal calcium transport.

Hint2 is expressed in the mitochondria of H295R cells and is involved in steroidogenesis

Hint2 belongs to the superfamily of histidine triad hydrolase enzymes. Recently, it has been shown to influence the mitochondria-dependent apoptosis occurring in hepatocytes, but its mechanism of action is still obscure. Here, we demonstrate that Hint2 is expressed in the mitochondria of H295R cells and in normal adrenals, and that this protein is involved in steroidogenesis. The presence of Hint2 in H295R cells was revealed by RT-PCR and by immunoblot analysis of subcellular fractions. The protein appeared associated with mitochondrial membranes, probably facing the interior of the organelle. Hint2 overexpression in H295R cells had no effect on pregnenolone secretion elicited by angiotensin II or K+, whereas protein silencing with specific small interfering RNA resulted in a marked reduction of the steroidogenic response. The duration of the mitochondrial calcium signal induced by angiotensin II was also reduced upon Hint2 down-regulation with small interfering RNA, but not affected after its overexpression, suggesting that under basal conditions, Hint2 is optimally expressed, and not rate limiting in steroidogenesis. Moreover, Hint2 also appeared involved in Ca2+-independent pathways leading to steroid formation. Indeed, pregnenolone formation in response to either forskolin or a hydroxyl analog of cholesterol was markedly reduced after Hint2 silencing. Calcium-dependent and calcium-independent actions of Hint2 on steroidogenesis could be related to its ability to maintain a favorable mitochondrial potential. In conclusion, these data suggest that, in H295R cells, Hint2 is required for an optimal steroidogenic response, possibly because of a particular signalling function exerted within the mitochondria and that still remains to determine at the molecular level.

Effect of once-yearly zoledronic acid 5 mg on fracture risk and change in femoral neck bone mineral density

Context: In the Health Outcomes and Reduced Incidence with Zoledronic Acid Once Yearly - Pivotal Fracture Trial (HORIZON-PFT), zoledronic acid (ZOL) 5 mg significantly reduced fracture risk. Objective: To identify factors associated with greater efficacy during ZOL 5 mg treatment. Design, Setting and Patients: Subgroup analysis (preplanned and post hoc) of a multicenter, double-blind, placebo-controlled, 36-month trial in 7765 women with postmenopausal osteoporosis. Intervention: Single infusion of ZOL 5 mg or placebo at baseline, 12 and 24 months. Main Outcome Measures: Primary endpoints: new vertebral fracture and hip fracture. Secondary endpoints: non-vertebral fracture, change in femoral neck bone mineral density (BMD). Baseline risk factor subgroups: age, BMD T-score and vertebral fracture status, total hip BMD, race, weight, geographical region, smoking, height loss, history of falls, physical activity, prior bisphosphonates, creatinine clearance, body mass index (BMI), concomitant osteoporosis medications. Results: Greater ZOL induced effects on vertebral fracture risk with younger age (treatment-by-subgroup interaction P=0.05), normal creatinine clearance (P=0.04), and BMI >/=25 kg/m(2) (P=0.02). There were no significant treatment-factor interactions for hip or non-vertebral fracture or for change in BMD. Conclusions: ZOL appeared more effective in preventing vertebral fracture in younger women, overweight/obese women and women with normal renal function. ZOL had similar effects irrespective of fracture risk factors or femoral neck BMD.

Glucagon-like peptide-1 receptor imaging for localization of insulinomas

The surgical removal of insulinomas is hampered by difficulties to localize it using conventional radiological procedures. Recently these tumors were shown to exhibit a very high density of glucagon-like peptide-1 receptors (GLP-1R) in vitro that may be used as specific targets for in vivo receptor radiolabeling.

Expanding the spectrum of mutations in GH1 and GHRHR: genetic screening in a large cohort of patients with congenital isolated growth hormone deficiency

CONTEXT: It is estimated that 3-30% of cases with isolated GH deficiency (IGHD) have a genetic etiology, with a number of mutations being reported in GH1 and GHRHR. The aim of our study was to genetically characterize a cohort of patients with congenital IGHD and analyze their characteristics. PATIENTS AND METHODS: A total of 224 patients (190 pedigrees) with IGHD and a eutopic posterior pituitary were screened for mutations in GH1 and GHRHR. To explore the possibility of an association of GH1 abnormalities with multiple pituitary hormone deficiencies, we have screened 62 patients with either multiple pituitary hormone deficiencies (42 pedigrees), or IGHD with an ectopic posterior pituitary (21 pedigrees). RESULTS: Mutations in GH1 and GHRHR were identified in 41 patients from 21 pedigrees (11.1%), with a higher prevalence in familial cases (38.6%). These included previously described and novel mutations in GH1 (C182X, G120V, R178H, IVS3+4nt, a>t) and GHRHR (W273S, R94L, R162W). Autosomal dominant, type II IGHD was the commonest form (52.4%), followed by type IB (42.8%) and type IA (4.8%). Patients with type II IGHD had highly variable phenotypes. There was no difference in the endocrinology or magnetic resonance imaging appearance between patients with and without mutations, although those with mutations presented with more significant growth failure (height, -4.7 +/- 1.6 SDS vs. -3.4 +/- 1.7 SDS) (P = 0.001). There was no apparent difference between patients with mutations in GH1 and GHRHR. CONCLUSIONS: IGHD patients with severe growth failure and a positive family history should be screened for genetic mutations; the evolving endocrinopathy observed in some of these patients suggests the need for long-term follow-up.

Mutation analysis of the muscarinic cholinergic receptor genes in isolated growth hormone deficiency type IB

BACKGROUND: Isolated GH deficiency (IGHD) is familial in 5-30% of patients. The most frequent form (IGHD-IB) has autosomal recessive inheritance, and it is known that it can be caused by mutations in the GHRH receptor (GHRHR) gene or in the GH gene. However, most forms of IGHD-IB have an unknown genetic cause. In normal subjects, muscarinic cholinergic stimulation causes an increase in pituitary GH release, whereas its blockade has the opposite effect, suggesting that a muscarinic acetylcholine receptor (mAchR) is involved in stimulating GH secretion. Five types of mAchR (M(1)-M(5)) exist. A transgenic mouse in which the function of the M(3) receptor was selectively ablated in the central nervous system has isolated GH deficiency similar to animals with defective GHRH or GHRHR gene. OBJECTIVE: We hypothesized that mAchR mutations may cause a subset of familial IGHD. PATIENTS/METHODS: After confirming the expression of M(1)-M(5) receptor mRNA in human hypothalamus, we analyzed the index cases of 39 families with IGHD-IB for mutations in the genes encoding for the five receptors. Coding sequences for each of the five mAchRs were subjected to direct sequencing. RESULTS: In one family, an affected member was homozygous for a M(3) change in codon 65 that replaces valine with isoleucine (V65I). The V65I receptor was expressed in CHO cells where it had normal ability to transmit methacholine signaling. CONCLUSION: mAchR mutations are absent or rare (less than 2.6%) in familial IGHD type IB.

Lethal respiratory failure and mild primary hypothyroidism in a term girl with a de novo heterozygous mutation in the TITF1/NKX2.1 gene

CONTEXT: Thyroid transcription factor 1 (TITF1/NKX2.1) is expressed in the thyroid, lung, ventral forebrain, and pituitary. In the lung, TITF1/NKX2.1 activates the expression of genes critical for lung development and function. Titf/Nkx2.1(-/-) mice have pituitary and thyroid aplasia but also impairment of pulmonary branching. Humans with heterozygous TITF1/NKX2.1 mutations present with various combinations of primary hypothyroidism, respiratory distress, and neurological disorders. OBJECTIVE: The objective of the study was to report clinical and molecular studies of the first patient with lethal neonatal respiratory distress from a novel heterozygous TITF1/NKX2.1 mutation. Participant: This girl, the first child of healthy nonconsanguineous French-Canadian parents, was born at 41 wk. Birth weight was 3,460 g and Apgar scores were normal. Soon after birth, she developed acute respiratory failure with pulmonary hypertension. At neonatal screening on the second day of life, TSH was 31 mU/liter (N <15) and total T(4) 245 nmol/liter (N = 120-350). Despite mechanical ventilation, thyroxine, surfactant, and pulmonary vasodilators, the patient died on the 40th day. RESULTS: Histopathology revealed pulmonary tissue with low alveolar counts. The thyroid was normal. Sequencing of the patient's lymphocyte DNA revealed a novel heterozygous TITF1/NKX2.1 mutation (I207F). This mutation was not found in either parent. In vitro, the mutant TITF-1 had reduced DNA binding and transactivation capacity. CONCLUSION: This is the first reported case of a heterozygous TITF1/NKX2.1 mutation leading to neonatal death from respiratory failure. The association of severe unexplained respiratory distress in a term neonate with mild primary hypothyroidism is the clue that led to the diagnosis.