Inactivating calcium-sensing receptor mutations in patients with primary hyperparathyroidism.

OBJECTIVE: Primary hyperparathyroidism (HPT) is characterised by autonomous secretion of PTH from enlarged parathyroid glands leading, in most patients, to asymptomatic hypercalcaemia. Familial hypocalciuric hypercalcaemia (FHH) is an autosomal dominant disorder caused by inactivating mutations in the calcium-sensing receptor (CaSR) gene; it is characterised by lifelong and usually asymptomatic hypercalcaemia. Establishing the correct diagnosis is important because surgery can be curative in HPT, but ineffective in FHH. There is overlap in the diagnostic criteria for the two disorders and some patients carrying inactivating mutations in the CaSR gene, which is suggestive of FHH, also have HPT with hyperplastic parathyroid glands or adenomas. DESIGN AND PATIENTS CaSR gene mutations were analysed and clinical and biochemical parameters evaluated in 139 consecutive outpatients presenting with hypercalcaemia and suspected of having HPT. RESULTS: Six different mutations of the CaSR gene were found in eight patients. In four patients, classical FHH was suspected based on clinical and biochemical results and was confirmed by the CaSR mutations. In the other four patients, HPT was diagnosed based on the biochemical profile or symptoms; in these four patients, the parathyroids were operated on and single adenomas were histologically confirmed. In all four patients, serum calcium decreased postoperatively; and in three patients, serum calcium normalised postoperatively. The CaSR mutations in these patients were R25X, E250K and Q926R. CONCLUSION: The coexistence of HPT and FHH in four of 139 patients suggests a pathogenetic role of CaSR mutations in HPT. Despite also having a CaSR mutation, these patients benefited from parathyroid surgery.

Activating mutations in the calcium-sensing receptor: genetic and clinical spectrum in 25 patients with autosomal dominant hypocalcaemia - a German survey.

OBJECTIVE: Autosomal dominant hypocalcaemia or hypoparathyroidism is caused by activating mutations of the calcium-sensing receptor (CaSR). Treatment with calcium and vitamin D often worsens hypercalciuria and nephrocalcinosis, and renal impairment can result. Our aim was to describe the phenotypic variance of this rare disorder in a large series and to evaluate the outcome after long-term treatment. DESIGN: Nationwide retrospective collaborative study. PATIENTS: We describe 25 patients (14 men and 11 women), 20 belonging to 11 families and five single cases. MEASUREMENTS: Activating CaSR mutations and clinical and biochemical findings were evaluated. RESULTS: Nine different missense mutations of the CaSR, including one novel variant (M734T), were found. Twelve patients (50%) were symptomatic, 9 (36%) had basal ganglia calcifications and 3 (12%) had nephrocalcinosis. Serum calcium was decreased (1·87 ± 0·13 mm), and PTH was decreased (n = 19) or inappropriately low (n = 4). The occurrence of hypocalcaemic symptoms at diagnosis was related to the degree of hypocalcaemia. The occurrence of features like calcification of basal ganglia or kidney calcification did not correlate with the severity of hypocalcaemia or the age at diagnosis. The most common treatment was calcitriol (median dosage 0·6 µg/day), and the mean duration of therapy was 7·1 years (max. 26 years). Hypercalcaemic episodes rarely occurred, and the rate of kidney calcifications was remarkably low (12%). CONCLUSION: This series increases the limited knowledge of mutations and phenotypes of this rare disorder. Mutation analysis of the CaSR gene facilitates patient and family management. Low dosages of calcitriol resulted in less frequent renal calcifications.

Novel inactivating mutations of the calcium-sensing receptor: the calcimimetic NPS R-568 improves signal transduction of mutant receptors.

CONTEXT AND OBJECTIVE: Inactivating mutations in the calcium-sensing receptor (CaSR) gene cause neonatal severe hyperparathyroidism and familial hypocalciuric hypercalcemia (FHH). The aims of the present study were the functional characterization of novel mutations of the CaSR found in FHH patients, the comparison of in vitro receptor function with clinical parameters, and the effect of the allosteric calcimimetic NPS R-568 on the signaling of mutant receptors. METHODS: Wild-type and mutant CaSRs (W530G, C568Y, W718X, M734R, L849P, Q926R, and D1005N) were expressed in human embryonic kidney 293 cells. Receptor signaling was studied by measuring intracellular free calcium in response to different concentrations of extracellular calcium ([Ca(2+)](o)). RESULTS: Four CaSR mutations (C568Y, W718X, M734R, and L849P) demonstrated a complete lack of a [Ca(2+)](o)-induced cytosolic Ca(2+) response up to 30 mm [Ca(2+)](o), whereas the CaSR mutants W530G, Q926R, and D1005N retained some sensitivity to [Ca(2+)](o). There was no significant relation between the in vitro calcium sensitivity, serum calcium, and intact PTH levels in the patients. Patients with C-terminal CaSR mutations had a calcium to creatine ratio above the established diagnostic threshold of 0.01 for FHH. The calcimimetic NPS R-568 enhanced the responsiveness to [Ca(2+)](o) in CaSR mutants of the extracellular domain (W530G and C568Y) as well as the intracellular C-terminal domain (Q926R and D1005N). CONCLUSION: Therefore, calcimimetics might offer medical treatment for symptomatic FHH patients, and more important, for patients with neonatal severe hyperparathyroidism that harbor calcimimetic-sensitive CaSR mutants.

Heterozygous inactivating CaSR mutations causing neonatal hyperparathyroidism: function, inheritance and phenotype.

BACKGROUND: Homozygous inactivating mutations of the calcium-sensing receptor (CaSR) lead to neonatal severe hyperparathyroidism (NSHPT), whereas heterozygous inactivating mutations result in familial hypocalciuric hypercalcemia (FHH). It is unknown why in some cases heterozygous CaSR mutations cause neonatal hyperparathyroidism (NHPT) clinically similar to NSHPT but with only moderately elevated serum calcium. METHODS: A literature survey was conducted to identify patients with heterozygous CaSR mutations and NHPT. The common NHPT CaSR mutants R185Q and R227L were compared with 15 mutants causing only FHH in the heterozygous state. We studied in vitro calcium signaling including the functional consequences of co-expression of mutant and wild-type (wt) CaSR, patients' phenotype, age of disease manifestation and mode of inheritance. RESULTS: All inactivating CaSR mutants impaired calcium signaling of wt-CaSR regardless of the patients' clinical phenotype. The absolute intracellular calcium signaling response to physiologic extracellular calcium concentrations in vitro showed a high correlation with patients' serum calcium concentrations in vivo, which is similar in NHPT and FHH patients with the same genotype. Pedigrees of FHH families revealed that paternal inheritance per se does not necessarily lead to NHPT but may only cause FHH. CONCLUSIONS: There is a significant correlation between in vitro functional impairment of the CaSR at physiologic calcium concentrations and the severity of alterations in calcium homeostasis in patients. Whether a particular genotype leads to NHPT or FHH appears to depend on additional predisposing genetic or environmental factors. An individual therapeutic approach appears to be warranted for NHPT patients.

Activation of the extraneuronal monoamine transporter (EMT) from rat expressed in 293 cells.

1. The extraneuronal monoamine transporter from rat (EMTr) was heterologously expressed by stable transfection in human embryonic kidney 293 cells and characterized in radiotracer experiments. 2. EMTr-mediated uptake of 1-methyl-4-phenylpyridinium (MPP(+)) was saturable, with a K(m) of 151 micro mol l(-1) and V(max) of 7.5 nmol min(-1) mg protein(-1). 3. Compared to the human orthologue EMTh (gene symbol SLC22A3), EMTr was about two orders of magnitude more resistant to most inhibitors, including disprocynium24 and corticosterone. 4. Strikingly, inhibitors and substrates at low concentration stimulated EMTr-mediated transport above control level with MPP(+) and noradrenaline as substrate, but not with cimetidine. Results were confirmed with EMT from mouse. 5. With different IC(50)-values for different substrates, the standard method to calculate K(i)-values is not applicable. 6. Our experiments suggest that activation is not caused by changes in membrane potential or trans-stimulation. Since the extent of activation depends markedly on the chemical structure of the monitored substrate, involvement of a receptor-mediated signalling pathway or recruitment of transporter reserve are implausible. 7. To explain activation, we present a kinetic model which assumes two binding sites for substrate or inhibitor per transporter entity, possibly resulting from the assembly of homodimers. 8. Activation explains previous reports about inhibitor-insensitive catecholamine transport in rat brain. 9. We speculate that activation may serve to keep the transporter working for specific substrates in the face of inhibitors.

Severe form of thyroid hormone resistance in a patient with homozygous/hemizygous mutation of T3 receptor gene.

Resistance to thyroid hormone syndrome (RTH) is a rare disorder, usually inherited as an autosomal dominant trait. Patients with RTH are usually euthyroid but can occasionally present with signs and symptoms of thyrotoxicosis or rarely with hypothyroidism. Affected individuals are usually heterozygous for mutations in the thyroid hormone receptor beta gene (TR-beta). We present a patient with RTH found to be homo-/hemizygous for a mutation in the TR-beta gene. The single nucleotide substitution I280S (1123T-->G) was present either on both alleles or in a hemizygous form with complete deletion of the second allele. The I280S mutation was recently reported in a heterozygous patient. The severe phenotype with seriously impaired intellectual development, hyperkinetic behaviour, tachycardia, hearing and visual impairment is probably due to the dominant negative effect of the I280S mutant protein and the absence of any functional TR-beta.

Amino alcohol- (NPS-2143) and quinazolinone-derived calcilytics (ATF936 and AXT914) differentially mitigate excessive signalling of calcium-sensing receptor mutants causing Bartter syndrome Type 5 and autosomal dominant hypocalcemia.

INTRODUCTION: Activating calcium sensing receptor (CaSR) mutations cause autosomal dominant hypocalcemia (ADH) characterized by low serum calcium, inappropriately low PTH and relative hypercalciuria. Four activating CaSR mutations cause additional renal wasting of sodium, chloride and other salts, a condition called Bartter syndrome (BS) type 5. Until today there is no specific medical treatment for BS type 5 and ADH. We investigated the effects of different allosteric CaSR antagonists (calcilytics) on activating CaSR mutants. METHODS: All 4 known mutations causing BS type 5 and five ADH mutations were expressed in HEK 293T cells and receptor signalling was studied by measurement of intracellular free calcium in response to extracellular calcium ([Ca2+]o). To investigate the effect of calcilytics, cells were stimulated with 3 mM [Ca2+]o in the presence or absence of NPS-2143, ATF936 or AXT914. RESULTS: All BS type 5 and ADH mutants showed enhanced signalling activity to [Ca2+]o with left shifted dose response curves. In contrast to the amino alcohol NPS-2143, which was only partially effective, the quinazolinone calcilytics ATF936 and AXT914 significantly mitigated excessive cytosolic calcium signalling of all BS type 5 and ADH mutants studied. When these mutants were co-expressed with wild-type CaSR to approximate heterozygosity in patients, ATF936 and AXT914 were also effective on all mutants. CONCLUSION: The calcilytics ATF936 and AXT914 are capable of attenuating enhanced cytosolic calcium signalling activity of CaSR mutations causing BS type 5 and ADH. Quinazolinone calcilytics might therefore offer a novel treatment option for patients with activating CaSR mutations.

Prevalence and clinical spectrum of nonsecretory medullary thyroid carcinoma in a series of 839 patients with sporadic medullary thyroid carcinoma.

BACKGROUND: Medullary thyroid carcinoma (MTC) is characterized by the synthesis and secretion of calcitonin (Ct). MTC without Ct secretion has been reported on rare occasions. The aim of this study was to analyze the prevalence and clinical spectrum of nonsecretory MTC in two tertiary centers that cared for 839 patients with sporadic MTC. METHODS: Clinical, biochemical, histological, and immunohistological findings, and somatic RET mutations were analyzed, and long-term follow-up was documented. RESULTS: Seven patients with nonsecretory MTC were identified among 839 patients with sporadic MTC; thus, the prevalence rate of nonsecretory MTC was 0.83%. In these seven patients, Ct and carcinoembryonic antigen (CEA) levels were normal when MTC was initially diagnosed in the patients, despite advanced tumor stage. Ct and CEA levels remained undetectable in four patients; recurrence was indicated in one patient after 10 years of follow-up by routine anatomic imaging and increased CEA levels, and Ct levels became slightly elevated during follow-up, despite massive tumor load, in the remaining two patients. The diagnosis of MTC was confirmed by positive immunohistochemistry for Ct, CEA, and chromogranin A. A high Ki67 proliferation index (PI) (three patients) and a high proportion of RET 918-mutated cells (four patients), as well as poorly differentiated histology, were associated with aggressive biological behavior of the MTC. The prognosis for nonsecretory MTC varied between long-term survival (12.5 years) and rapid progression leading to death within 1.75 years after diagnosis. CONCLUSIONS: The prevalence of nonsecretory MTC was low (0.83% of patients with MTC). Diagnosis was often made at a clinically advanced tumor stage. The histological and immunohistological characteristics and the clinical course and prognosis of nonsecretory MTC are markedly heterogeneous. A high Ki-67 PI and a large proportion of cells with RET 918 mutations are associated with a poor prognosis.

Diagnosis of glucocorticoid-remediable aldosteronism in hypertensive children.

OBJECTIVE: Glucocorticoid-remediable aldosteronism (GRA) is caused by the presence of a chimeric gene originating from an unequal cross-over between the CYP11B1 and CYP11B2 genes. Aldosterone suppression by dexamethasone and high 18-hydroxycortisol (18-OHF) levels have been used to differentiate GRA from the other forms of primary aldosteronism. METHODS: A dexamethasone suppression test including serum 18-OHF determination and the measurement of urinary excretion of aldosterone, its metabolites and 18-OHF were performed in 3 children of a family with primary aldosteronism. Polymerase chain reactions were performed to identify the chimeric gene. RESULTS: The chimeric gene was identified in 2 children, their mother and grandmother. The affected children had an aldosterone-to-plasma renin activity ratio >30, elevated serum 18-OHF concentration and increased urinary excretion of aldosterone, its metabolites, and 18-OHF. Post-dexamethasone concentrations of serum aldosterone and 18-OHF concentrations were suppressed. CONCLUSION: Although very rare, the possible diagnosis of GRA should be considered in all children or young adults with low-renin hypertension. Since genetic testing is more specific than biochemical testing, a definitive diagnosis can only be obtained by identification of the CYP11B1/CYP11B2 chimeric gene.

CDC73-related hereditary hyperparathyroidism: five new mutations and the clinical spectrum.

OBJECTIVE: Hyperparathyroidism-jaw tumour (HPT-JT) syndrome is a rare autosomal dominant cause of benign and malignant parathyroid tumours, ossifying jaw tumours, various cystic and neoplastic renal abnormalities and benign and malignant uterine tumours. Disease-causing mutations have been localised in the tumour suppressor gene CDC73. There is limited information available on the mutations, and resulting phenotypes and long-term follow-up data are especially scarce. DESIGN: We analysed the clinical data from 16 patients (including three families) carrying mutations in the CDC73 gene. We describe five new mutations/gene variants, the corresponding phenotypes of these carriers and the long-term follow-up. METHODS: The 16 patients were evaluated at an endocrine outpatient clinic and at a surgical department. DNA samples were obtained for sequence analysis of the CDC73 gene. RESULTS: Clinical features of HPT-JT syndrome were detected in 13 of the 15 carriers with germline CDC73 mutations. The major features were benign (n=7; 47%) or cancerous (n=3; 20%) HPT-JT was present in eight cases (53%). Most patients had severe hypercalcaemia, and median serum calcium levels were 3.36 mmol/l. A patient with non-secretory parathyroid carcinoma was included. HPT was diagnosed at a median age of 28.5 years. Mutational analysis of the CDC73 gene identified eight sequence changes, three of them have been reported previously, whereas five are novel: c.1346delG, c.88_94delTTCTCCT, the non-coding variants, c.307+5G>T and c.424-5T>C and c.*12C>A of unknown significance. CONCLUSIONS: This study significantly increases the information available on the mutations and phenotypes of HPT-JT syndrome.

Novel activating mutations of the calcium-sensing receptor: the calcilytic NPS-2143 mitigates excessive signal transduction of mutant receptors.

CONTEXT AND OBJECTIVE: Activating mutations in the calcium-sensing receptor (CaSR) gene cause autosomal dominant hypocalcemia (ADH). The aims of the present study were the functional characterization of novel mutations of the CaSR found in patients, the comparison of in vitro receptor function with clinical parameters, and the effect of the allosteric calcilytic NPS-2143 on the signaling of mutant receptors as a potential new treatment for ADH patients. METHODS: Wild-type and mutant CaSR (T151R, P221L, E767Q, G830S, and A844T) were expressed in human embryonic kidney cells (HEK 293T). Receptor signaling was studied by measuring intracellular free calcium in response to different concentrations of extracellular calcium ([Ca(2+)](o)) in the presence or absence of NPS-2143. RESULTS: All ADH patients had lowered serum calcium ranging from 1.7 to 2.0 mm and inadequate intact PTH and urinary calcium excretion. In vitro testing of CaSR mutations from these patients revealed exaggerated [Ca(2+)](o)-induced cytosolic Ca(2+) responses with EC(50) values for [Ca(2+)](o) ranging from 1.56 to 3.15 mM, which was lower than for the wild-type receptor (4.27 mM). The calcilytic NPS-2143 diminished the responsiveness to [Ca(2+)](o) in the CaSR mutants T151R, E767Q, G830S, and A844T. The mutant P221L, however, was only responsive when coexpressed with the wild-type CaSR. CONCLUSION: Calcilytics might offer medical treatment for patients with autosomal dominant hypocalcemia caused by calcilytic-sensitive CaSR mutants.

[Diagnostic procedure and antidementive therapy of dementia patients in primary care: an urban-rural comparison]. / Diagnostik und Therapie von Demenzerkrankungen in der hausärztlichen Praxis: ein Stadt-Land-Vergleich.

OBJECTIVES: The aim of this work was to investigate the diagnostic and therapeutic regime of general practitioners (GPs) of dementia patients in an urban-rural-comparison. METHODS: The cross-sectional analysis of 129 GPs and 390 patients is part of the three-arm cluster-randomized controlled follow-up-study Project IDA - Initiative dementia care in primary practice . The study was carried out in Middle Franconia, a typical urban-rural area in Southeastern Germany. The sample constitutes the baseline assessment. GPs had to document their diagnostic and therapeutic behaviour upon inclusion of dementia patients in the study. RESULTS: As expected, significant differences in imaging diagnostic procedures exist between urban (57.9 %) and rural (42.7 %) areas (p = 0.010). There are no differences in the referral quote to specialists (total 57.9 %), in the frequency of treatment with non-pharmacological therapies (total 13.3 %) as well as pharmacological therapies with evidence-based medication (18.7 %; AChE inhibitors and Memantine). CONCLUSIONS: Except for imaging diagnostic procedures, there were no differences in the regime of urban and rural GPs. Thus, an almost similar care for rural and urban dementia patients can be assumed.

Screening for Pax8 mutations in patients with congenital hypothyroidism in South-West Germany.

AIMS: To study the frequency of mutations in the Pax8 gene in a cohort of patients with congenital hypothyroidism (CH) in South West Germany. METHODS: A cohort of 95 patients with CH (60 females, 35 males), identified in our newborn screening program, was analyzed for mutations in Pax8 by single-stranded conformational polymorphism (SSCP) and DNA sequencing. RESULTS: SSCP analysis and direct sequencing of exon 3 of a female patient with a hypoplastic thyroid gland revealed two heterozygous mutations in Pax8 resulting in a transition of T to C (codon 34) and G to A (codon 35), replacing isoleucine by threonine and valine by isoleucine. Using allele-specific PCR we could demonstrate that both mutations are located on the same allele. Furthermore, a polymorphism was documented in 24 patients with thyroid hypoplasia in intron 6 at nucleotide +51 (CC, GG, CG). Comparison of the polymorphisms between hypothyroid patients and controls revealed no significant differences suggesting that this polymorphism does not play a role in the pathogenesis of hypothyroidism. No further mutations or polymorphisms were found in the cohort. CONCLUSIONS: These findings confirm the contribution of mutations in the Pax8 gene to the etiology of thyroid dysgenesis with a variable penetrance, but also demonstrate the rare overall incidence in CH.

The localisation of the extraneuronal monoamine transporter (EMT) in rat brain.

The extraneuronal monoamine transporter plays an important role in the inactivation of monoamine transmitters. A basal extraneuronal tissue expression of this transporter has been reported, but it is also expressed in CNS glia. As little is known about the expression pattern and the function of the extraneuronal monoamine transporter in the brain, we performed a detailed investigation. Firstly, a northern blot analysis of different rat organs revealed that the transporter is strongly expressed in placenta, lung and heart and less prominently in the whole brain, brain stem, intestine, testis, epididymis, stomach, kidney and skeletal muscle. It was not expressed in cerebellum, liver and embryo. Using an in situ hybridization to the rat brain, we detected a marked and highly confined expression of the extraneuronal monoamine transporter in the area postrema, but in no other brain areas. These findings were confirmed by polyclonal antibodies against rat extraneuronal monoamine transporter showing an intensive signal in the area postrema, although a few cells in the cerebellum and the brain stem also showed a signal. Additionally, a partly overlapping expression pattern of the monoamine oxidase-B was detected. Summarizing, we firstly describe a marked and highly confined expression of the extraneuronal monoamine transporter in the rat area postrema by in situ hybridisation which may play a role in physiological functions of this circumventricular organ such as emesis, food intake and the regulation of cardiovascular functions.