The calcilytic agent NPS 2143 rectifies hypocalcemia in a mouse model with an activating calcium-sensing-receptor (CaSR) mutation: relevance to autosomal dominant hypocalcemia type 1 (ADH1) (original) (raw)
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Gα11 mutation in mice causes hypocalcemia rectifiable by calcilytic therapy
JCI insight, 2017
Heterozygous germline gain-of-function mutations of G-protein subunit α11 (Gα11), a signaling partner for the calcium-sensing receptor (CaSR), result in autosomal dominant hypocalcemia type 2 (ADH2). ADH2 may cause symptomatic hypocalcemia with low circulating parathyroid hormone (PTH) concentrations. Effective therapies for ADH2 are currently not available, and a mouse model for ADH2 would help in assessment of potential therapies. We hypothesized that a previously reported dark skin mouse mutant (Dsk7) - which has a germline hypermorphic Gα11 mutation, Ile62Val - may be a model for ADH2 and allow evaluation of calcilytics, which are CaSR negative allosteric modulators, as a targeted therapy for this disorder. Mutant Dsk7/+ and Dsk7/Dsk7 mice were shown to have hypocalcemia and reduced plasma PTH concentrations, similar to ADH2 patients. In vitro studies showed the mutant Val62 Gα11 to upregulate CaSR-mediated intracellular calcium and MAPK signaling, consistent with a gain of func...
The Journal of Clinical Endocrinology and Metabolism, 2008
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 2ϩ ͔ o). Results: Four CaSR mutations (C568Y, W718X, M734R, and L849P) demonstrated a complete lack of a ͓Ca 2ϩ ͔ o-induced cytosolic Ca 2ϩ response up to 30 mM ͓Ca 2ϩ ͔ o , whereas the CaSR mutants W530G, Q926R, and D1005N retained some sensitivity to ͓Ca 2ϩ ͔ 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 2ϩ ͔ 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.
European Journal of Endocrinology, 2015
The calcium-sensing receptor (CASR) is the main calcium sensor in the maintenance of calcium metabolism. Mutations of the CASR, the G protein alpha 11 (GNA11) and the adaptor-related protein complex 2 sigma 1 subunit (AP2S1) genes can shift the set point for calcium sensing causing hyper- or hypo-calcemic disorders. Therapeutic concepts for these rare diseases range from general therapies of hyper- and hypo-calcemic conditions to more pathophysiology oriented approaches such as parathyroid hormone (PTH) substitution and allosteric CASR modulators. Cinacalcet is a calcimimetic that enhances receptor function and has gained approval for the treatment of hyperparathyroidism. Calcilytics in turn attenuate CASR activity and are currently under investigation for the treatment of various diseases. We conducted a literature search for reports about treatment of patients harboring inactivating or activating CASR, GNA11 or AP2S1 mutants and about in vitro effects of allosteric CASR modulators...
Autosomal dominant hypocalcemia due to a truncation in the C-tail of the calcium-sensing receptor
Molecular and cellular endocrinology, 2016
Autosomal Dominant Hypocalcemia (ADH) is an endocrine disorder due to activating mutations of the calcium-sensing receptor (CASR) gene. We report on a young boy who presented low serum calcium with hypercalciuria, hyperphosphatemia and low serum concentration of parathyroid hormone, not accompanied by classic clinical signs of hypocalcemia. Treatment with calcitriol and calcium did not normalize serum calcium and renal calcium excretion. The use of thiazide diuretics slightly reduced calciuria. Despite high calcium excretion, no signs of nephrocalcinosis were detected. The patient had a prolonged Q-T interval at ECG, which did not normalize during treatment. PCR amplification of CASR coding sequence and direct sequencing of PCR products. showed a novel heterozygous deletion of a cytosine (c.2682delC), responsible for a frameshift (p.S895Pfs*44) and a premature stop codon resulting in a truncation of the CaSR's C-tail. Functional studies indicated increased activity of mutant rec...
The Journal of Clinical Endocrinology & Metabolism, 2013
Objective: Activating mutations in the calcium-sensing receptor (CASR) gene cause autosomal dominant hypoparathyroidism, and heterozygous inactivating CASR mutations cause familial hypocalciuric hypercalcemia. Recently, there has been a focus on the use of allosteric modulators to restore the functional activity of mutant CASRs. In this study, the effect of allosteric modulators NPS R-568 and NPS 2143 on CASR mutants was studied in vitro. Methods: DNA sequence analysis of the CASR gene was undertaken in autosomal dominant hypoparathyroidism and familial hypocalciuric hypercalcemia Japanese patients, and the functional consequences for the Gi-MAPK pathway and cell surface expression of CASR were determined. Furthermore, we studied the effect of NPS R-568 and NPS 2143 on the signal transduction activity and cell surface expression of each mutant CASR. Results: We identified 3 activating mutations (S122C, P569H, and I839T) and 2 inactivating mutations (A110T and R172G) in patients. The...
CASR gene activating mutations in two families with autosomal dominant hypocalcemia
Molecular Genetics and Metabolism, 2012
Background: Autosomal dominant hypocalcemia (ADH) is an endocrine disorder caused by activating mutations of the calcium-sensing receptor (CASR) gene which plays a major role in maintaining calcium homeostasis. Biochemical features of ADH are hypocalcemia and hypercalciuria with inappropriately low levels of parathyroid hormone (PTH). We report on two four-generation families affected by ADH. Aim: To identify mutations of CASR gene in subjects affected by familial idiopathic hypoparathyroidism. To perform functional assays of identified CASR variants by transient transfection on HEK293 cells. Results: We identified two CASR variants (Q681R and P221L): the Q681R variant was novel while the P221L had been previously published. Functional assays on the Q681R variant showed that it did not alter the whole expression nor the correct plasmamembrane localization, but enhanced the signaling function, increasing the sensitivity of the receptor as compared to the WT. Conclusions: We report two activating CASR mutations in two families affected by ADH and the functional assays performed on the novel variant Q681R. Our work enlarged the spectrum of mutations of the CASR and contributed to a better elucidation of the protein function.
A novel mutation in calcium-sensing receptor gene associated to hypercalcemia and hypercalciuria
BMC Endocrine Disorders, 2014
Background: Familial Hyperparathyroidism (HPT) and Familial benign Hypocalciuric Hypercalcemia (FHH) are the most common causes of hereditary hypercalcemia. FHH has been demonstrated to be caused by inactivating mutations of calcium-sensing receptor (CaSR) gene, involved in PTH regulation as well as in renal calcium excretion. Case presentation: In two individuals, father and son, we found a novel heterozygous mutation in CaSR gene. The hypercalcemia was present only in father, which, by contrast to the classic form of FHH showed hypercalciuria (from 300 to 600 mg/24 h in different evaluations) and a Calcium/Creatinine ratio of 0.031, instead of low or normal calciuria (<0.01 typical finding in FHH). His son showed the same mutation in CaSR gene, but no clinical signs or hypercalcemia although serum ionized calcium levels were close to the upper limit of normal values (1.30 mmol/L: normal range: 1.12-1.31 mmol/L). Sequence analysis revealed a point mutation at codon 972 of CaSR gene (chromosome 3q), located within cytoplasmic domain of the CaSR, that changes Threonine with Methionine. The father was treated with Cinacalcet 90 mg/day, with a decrease of total serum calcemia from an average value of 12.2 mg/dl to 10.9 mg/dl.
PTH-independent regulation of blood calcium concentration by the calcium-sensing receptor
Journal of Clinical Investigation, 2012
Tight regulation of calcium levels is required for many critical biological functions. The Ca 2+-sensing receptor (CaSR) expressed by parathyroid cells controls blood calcium concentration by regulating parathyroid hormone (PTH) secretion. However, CaSR is also expressed in other organs, such as the kidney, but the importance of extraparathyroid CaSR in calcium metabolism remains unknown. Here, we investigated the role of extraparathyroid CaSR using thyroparathyroidectomized, PTH-supplemented rats. Chronic inhibition of CaSR selectively increased renal tubular calcium absorption and blood calcium concentration independent of PTH secretion change and without altering intestinal calcium absorption. CaSR inhibition increased blood calcium concentration in animals pretreated with a bisphosphonate, indicating that the increase did not result from release of bone calcium. Kidney CaSR was expressed primarily in the thick ascending limb of the loop of Henle (TAL). As measured by in vitro microperfusion of cortical TAL, CaSR inhibitors increased calcium reabsorption and paracellular pathway permeability but did not change NaCl reabsorption. We conclude that CaSR is a direct determinant of blood calcium concentration, independent of PTH, and modulates renal tubular calcium transport in the TAL via the permeability of the paracellular pathway. These findings suggest that CaSR inhibitors may provide a new specific treatment for disorders related to impaired PTH secretion, such as primary hypoparathyroidism.