Calcium-sensing Receptor Mutations Research Articles

A HomozygousCaSRMutation Causing a FHH Phenotype Completely Masked by Vitamin D Deficiency Presenting as Rickets

Heterozygous inactivating calcium-sensing receptor (CaSR) mutations lead to familial hypocalciuric hypercalcemia (FHH), whereas homozygous mutations usually cause neonatal severe hyperparathyroidism. The objective of the study was to investigate the pathophysiological mechanisms of a homozygous inactivating CaSR mutation identified in a 16-year-old female. Clinical, biochemical, and genetic analyses of the index patient and her family were performed. Functional capacity of CaSRQ459R and CaSR mutants causing FHH (Q27R, P39A, S417C) or neonatal severe hyperparathyroidism (W718X) was assessed. Activation of the cytosolic calcium pathway and inhibition of PTH-induced cAMP signaling were measured. A 16-year-old girl presented with adolescent rickets, vitamin D deficiency, and secondary hyperparathyroidism. Vitamin D treatment unmasked features resembling FHH, and genetic testing revealed a homozygous CaSRQ459R mutation. Two apparently healthy siblings were homozygous for CaSRQ459R and had asymptomatic hypercalcemia and hypocalciuria. The CaSRQ459R mutation leads to mild functional inactivation in vitro, which explains the FHH-like phenotype in homozygous family members and the grossly exaggerated PTH response to vitamin D deficiency in the index case. The patient's parents and two other siblings were heterozygous, had normal serum calcium and PTH, but had marked hypocalciuria, which appeared to be associated with impaired in vitro activation of the calcium signaling pathway by CaSRQ459R. The Q459R mutation responded well to calcimimetic treatment in vitro. CaSR mutations causing mild functional impairment can lead to FHH, even in homozygous patients. The skeletal deformities in the index case were mainly due to severe vitamin D deficiency, and the CaSR mutation did not appear to have played a major independent role in the skeletal phenotype.

Pregnancy outcome in women with autosomal dominant hypocalcaemic hypercalciuric nephrocalcinosis

Autosomal dominant hypocalcaemic hypercalciuric nephrocalcinosis is an extremely rare clinical condition caused by an activating mutation of calcium-sensing receptor. Patients presenting with this condition are generally asymptomatic of hypocalcaemia inspite of significant lower serum calcium levels. Attempts at administering vitamin D to correct their hypocalcaemia tend to result in hypercalciuria with its attendant complications of nephrocalcinosis and renal impairment. To our knowledge there are no reports of pregnancy outcomes in women suffering with this ailment, hence this report of three pregnancies in two such women. In view of the rarity of this condition we hope it will assist professionals managing such cases.

G protein-coupled receptor mutations and human genetic disease.

Genetic variations in G protein-coupled receptor genes (GPCRs) disrupt GPCR function in a wide variety of human genetic diseases. In vitro strategies and animal models have been used to identify the molecular pathologies underlying naturally occurring GPCR mutations. Inactive, overactive, or constitutively active receptors have been identified that result in pathology. These receptor variants may alter ligand binding, G protein coupling, receptor desensitization and receptor recycling. Receptor systems discussed include rhodopsin, thyrotropin, parathyroid hormone, melanocortin, follicle-stimulating hormone (FSH), luteinizing hormone, gonadotropin-releasing hormone (GNRHR), adrenocorticotropic hormone, vasopressin, endothelin-β, purinergic, and the G protein associated with asthma (GPRA or neuropeptide S receptor 1 (NPSR1)). The role of activating and inactivating calcium-sensing receptor (CaSR) mutations is discussed in detail with respect to familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalemia (ADH). The CASR mutations have been associated with epilepsy. Diseases caused by the genetic disruption of GPCR functions are discussed in the context of their potential to be selectively targeted by drugs that rescue altered receptors. Examples of drugs developed as a result of targeting GPCRs mutated in disease include: calcimimetics and calcilytics, therapeutics targeting melanocortin receptors in obesity, interventions that alter GNRHR loss from the cell surface in idiopathic hypogonadotropic hypogonadism and novel drugs that might rescue the P2RY12 receptor congenital bleeding phenotype. De-orphanization projects have identified novel disease-associated receptors, such as NPSR1 and GPR35. The identification of variants in these receptors provides genetic reagents useful in drug screens. Discussion of the variety of GPCRs that are disrupted in monogenic Mendelian disorders provides the basis for examining the significance of common pharmacogenetic variants.

An interesting case of primary hypoparathyroidism

Primary hypoparathyroidism can occur due to an activating mutation of calcium sensing receptor (CaSR). Most patients remain asymptomatic and therefore not diagnosed until adulthood. We present a 38-year-old lady who had a history of muscle cramps since 8 years. She presented with vomiting, abdomen pain and body ache, showed clinical evidence of hypovolemia, severe hypocalcemia, hypokalemia, hypomagnesemia, hyperphosphatemia and metabolic alkalosis. Her 24 h urinary phosphorus was low and 24 h urinary excretion of sodium, potassium and chloride were high. Her intact parathormone was on the lower side of the normal range. She improved once we had corrected her biochemical abnormalities. By excluding acquired causes of hypoparathyroidism, we are able to conclude that this may be a case of primary hypoparathyroidism due to activating mutation of CaSR.

Ruling in a Suspect: The Role ofAP2S1Mutations in Familial Hypocalciuric Hypercalcemia Type 3

Ruling in a Suspect: The Role of<i>AP2S1</i>Mutations in Familial Hypocalciuric Hypercalcemia Type 3

Calcium-sensing receptor inhibits TGF-β-signaling by decreasing Smad2 phosphorylation

Calcium-sensing receptor (CaSR) contributes to maintain homeostatic levels of extracellular calcium. In addition, CaSR controls other cellular activities such as proliferation and migration, particularly in cells not related to extracellular calcium homeostasis, potentially by cross-talking with parallel signaling pathways. Here we report that CaSR attenuates transforming growth factor-β (TGF-β)-signaling in hepatic C9 cells and in transfected HEK293 cells. Wild type CaSR interferes with TGF-β-dependent Smad2 phosphorylation and induces its proteasomal degradation, resulting in a decrease of TGF-β-dependent transcriptional activity, whereas an inactivating CaSR mutant does not transduce an inhibitory effect of extracellular calcium on TGF-β signaling. Attenuation of TGF-β signaling in response to extracellular calcium is linked to Rab11-dependent CaSR-trafficking with the intervention of CaSR carboxyl-terminal tail. Our data suggest that CaSR might regulate TGF-β-dependent cellular responses mediated by TGF-β signaling inhibition.

Loss-of-Function and Gain-of-Function Mutations of Calcium-Sensing Receptor: Functional Analysis and the Effect of Allosteric Modulators NPS R-568 and NPS 2143

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. 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. We identified 3 activating mutations (S122C, P569H, and I839T) and 2 inactivating mutations (A110T and R172G) in patients. The activating and inactivating mutations caused leftward and rightward shifts, respectively, in the dose-response curves of the signaling pathway. NPS R-568 rescued the signal transduction capacity of 2 inactivating mutants without increasing cell surface expression levels. NPS 2143 suppressed the enhanced activity of the activating mutants without altering cell surface expression levels, although A843E, which is a constitutively active mutant, was suppressed to a lesser degree. We have identified 4 novel mutations of CASR. Moreover, our results indicate that allosteric modulators can restore the activity of the loss- and gain-of-function mutant CASRs, identified in this study.

Familial isolated primary hyperparathyroidism due to HRPT2 mutation

Primary hyperparathyroidism is a common endocrine disorder that is mostly caused by solitary tumors within the parathyroid glands. Characterized by early debut and higher frequency of multiple parathyroid masses, familial forms of primary hyperparathyroidism are caused by the already known mutations of: menin (MEN1 syndrome), RET proto-oncogene (MEN2 syndrome), HRPT2-parafibromin (hyperparathyroidism-jaw tumor syndrome), calcium sensing receptor gene (familial hypocalciuric hypercalcemia). A specific mutation in FIHP has not been identified in the majority of affected families. Recent studies revealed menin, HRPT2 and calcium-sensing receptor mutations in patients with FIHP. Whether FIHP is a variant or an early stage of MEN1 syndrome or hyperparathyroidism-jaw tumor syndrome is yet to be established. We present three siblings with familial isolated hyperparathyroidism due to solitary parathyroid adenoma and favorable evolution post-parathyroidectomy. Genetic tests revealed HRPT2 mutation.

Mutations Affecting G-Protein Subunit α11 in Hypercalcemia and Hypocalcemia

BackgroundFamilial hypocalciuric hypercalcemia is a genetically heterogeneous disorder with three variants: types 1, 2, and 3. Type 1 is due to loss-of-function mutations of the calcium-sensing receptor, a guanine nucleotide–binding protein (G-protein)–coupled receptor that signals through the G-protein subunit α11 (Gα11). Type 3 is associated with adaptor-related protein complex 2, sigma 1 subunit (AP2S1) mutations, which result in altered calcium-sensing receptor endocytosis. We hypothesized that type 2 is due to mutations effecting Gα11 loss of function, since Gα11 is involved in calcium-sensing receptor signaling, and its gene (GNA11) and the type 2 locus are colocalized on chromosome 19p13.3. We also postulated that mutations effecting Gα11 gain of function, like the mutations effecting calcium-sensing receptor gain of function that cause autosomal dominant hypocalcemia type 1, may lead to hypocalcemia.MethodsWe performed GNA11 mutational analysis in a kindred with familial hypocalciuric hypercalcemia type 2 and in nine unrelated patients with familial hypocalciuric hypercalcemia who did not have mutations in the gene encoding the calcium-sensing receptor (CASR) or AP2S1. We also performed this analysis in eight unrelated patients with hypocalcemia who did not have CASR mutations. In addition, we studied the effects of GNA11 mutations on Gα11 protein structure and calcium-sensing receptor signaling in human embryonic kidney 293 (HEK293) cells.ResultsThe kindred with familial hypocalciuric hypercalcemia type 2 had an in-frame deletion of a conserved Gα11 isoleucine (Ile200del), and one of the nine unrelated patients with familial hypocalciuric hypercalcemia had a missense GNA11 mutation (Leu135Gln). Missense GNA11 mutations (Arg181Gln and Phe341Leu) were detected in two unrelated patients with hypocalcemia; they were therefore identified as having autosomal dominant hypocalcemia type 2. All four GNA11 mutations predicted disrupted protein structures, and assessment on the basis of in vitro expression showed that familial hypocalciuric hypercalcemia type 2–associated mutations decreased the sensitivity of cells expressing calcium-sensing receptors to changes in extracellular calcium concentrations, whereas autosomal dominant hypocalcemia type 2–associated mutations increased cell sensitivity.ConclusionsGα11 mutants with loss of function cause familial hypocalciuric hypercalcemia type 2, and Gα11 mutants with gain of function cause a clinical disorder designated as autosomal dominant hypocalcemia type 2. (Funded by the United Kingdom Medical Research Council and others.)

Calcium homeostasis, and clinical or subclinical vitamin D deficiency – Can a hypothesis of “intestinal calcistat” explain it all?

The main physiological function of vitamin D is maintenance of calcium homeostasis by its effect on calcium absorption, and bone health in association with parathyroid gland. Vitamin D deficiency (VDD) is defined as serum 25-hydroxy vitamin D (25OHD) levels <20ng/ml. Vitamin D insufficiency is called when serum 25OHD levels are between 20–29ng/ml, though existence of this entity has been questioned. Do all subjects with VDD have clinical disease according to this definition? Analysis of published studies suggests that calcium absorption in inversely correlated with serum 25OHD levels and calcium intake. We hypothesize that there exist an intestinal calcistat, which controls the calcium absorption independent of PTH levels. It consists of calcium sensing receptor (CaSR) on intestinal brush border, which senses calcium in intestinal cells and vitamin D system in intestinal cells. CaSR dampens the generation of active vitamin D metabolite in intestinal cells and decrease active transcellular calcium transport. It also facilitates passive paracellular diffusion of calcium in intestine. This local adaptation adjusts the fractional calcium absorption according the body requirement. Failure of local adaptation due to decreased calcium intake, decreased supply of 25OHD, mutation in CaSR or vitamin D system decreases systemic calcium levels and systemic adaptations comes into the play. Systemic adaptations consist of rise in PTH and increase in active vitamin D metabolites. These adaptations lead to bone resorption and maintenance of calcium homeostasis. Not all subjects with varying levels of VDD manifest with secondary hyperparathyroidism and decreased in bone mineral density. We suggest that rise in PTH is first indicator of VDD is rise in PTH along with decrease in BMD depending on duration of VDD. Hence, subjects with any degree of VDD with normal PTH and BMD should not be labeled as vitamin D deficient. These subjects can be called subclinical VDD, and further studies are required to assess beneficial effect of vitamin D supplementation in this subset of population. This hypothesis further highlights pitfalls in treatment of hypoparathyroidism.

Molecular and clinical analysis of a neonatal severe hyperparathyroidism case caused by a stop mutation in the calcium-sensing receptor extracellular domain representing in effect a human ‘knockout’

Loss-of-function calcium-sensing receptor (CAR) mutations cause elevated parathyroid hormone (PTH) secretion and hypercalcaemia. Although full Car deletion is possible in mice, most human CAR mutations result from a single amino acid substitution that maintains partial function. However, here, we report a case of neonatal severe hyperparathyroidism (NSHPT) in which the truncated CaR lacks any transmembrane domain (CaR(R392X)), in effect a full CAR 'knockout'. The infant (daughter of distant cousins) presented with hypercalcaemia (5.5-6 mmol/l corrected calcium (2.15-2.65)) and elevated PTH concentrations (650-950 pmol/l (12-81)) together with skeletal demineralisation. NSHPT was confirmed by CAR gene sequencing (homozygous c.1174C-to-T mutation) requiring total parathyroidectomy during which only two glands were located and removed, resulting in normalisation of her serum PTH/calcium levels. The R392X stop codon was inserted into human CAR and the resulting mutant (CaR(R392X)) expressed transiently in HEK-293 cells. CaR(R392X) expressed as a 54 kDa dimeric glycoprotein that was undetectable in conditioned medium or in the patient's urine. The membrane localisation observed for wild-type CaR in parathyroid gland and transfected HEK-293 cells was absent from the proband's parathyroid gland and from CaR(R392X)-transfected cells. Expression of the mutant was localised to endoplasmic reticulum consistent with its lack of functional activity. Intriguingly, the patient remained normocalcaemic throughout childhood (2.5 mM corrected calcium, 11 pg/ml PTH (10-71), age 8 years) but exhibited mild asymptomatic hypocalcaemia at age 10 years, now treated with 1-hydroxycholecalciferol and Ca2+ supplementation. Despite representing a virtual CAR knockout, the patient displays no obvious pathologies beyond her calcium homeostatic dysfunction.

Identification and characterization of D410E, a novel mutation in the loop 3 domain of CASR, in autosomal dominant hypocalcemia and a therapeutic approach using a novel calcilytic, AXT914

Activating mutations of the calcium-sensing receptor (CASR) gene are associated with autosomal dominant hypocalcemia (ADH) characterized by benign hypocalcemia, inappropriately low (PTH) levels and mostly hypercalciuria. Herein, we report a novel activating mutation in the CASR gene in a Korean family with ADH. The CASR gene was sequenced in the patient with ADH. The identified mutations were also evaluated in the patient's family members by PCR-based sequencing. For functional studies, we examined phosphorylation of ERK1/2. In addition, intracellular Ca(2+) mobilization and the effects of the calcilytic, AXT914 were measured using fluorophore Fura-2 dye. Direct sequencing analysis of the CASR gene showed that the proband and her daughter possess a novel mutation c.1230T>A, resulting in a D410E missense mutation on exon 4 of the CASR gene. Escalation of the extracellular Ca(2+) concentration resulted in stronger phosphorylation of ERK1/2 and higher levels of intracellular Ca(2+) in HEK293 cells expressing mutant CASR, compared with wild-type CASR. The increase in intracellular Ca(2+) signalling via CASR was successively blunted by treatment with AXT914. Over 60 activating mutations in the CASR gene have been identified to cause ADH so far. Here, we add one more activating mutation that causes ADH. The novel activating mutation (D410E) occurred in the loop 3 region of CASR, where its function was believed to be of little importance; therefore, this mutation may be of interest. Further clinical study will be needed to validate the effectiveness of calcilytics in treatment of ADH in vivo.

Activating mutations of the calcium-sensing receptor: The calcilytics ATF-936 and AXT-914 attenuate mutants causing autosomal dominant hypocalcemia and Bartter syndrome type 5

Introduction: Activating mutations of the calcium sensing receptor (CaSR) 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, potassium and other salts, a condition called Bartter syndrome type 5. Until today there is no medical treatment for Bartter Syndrome and ADH. We therefore investigated the effects of two novel calcilytics on activating CaSR mutants.

Activating mutations of the calcium-sensing receptor: Calcium oscillations and negative feedback via PKC in mutants causing autosomal dominant hypocalcemia and Bartter syndrome type 5

Introduction: Activating mutations of the calcium sensing receptor (CaSR) cause autosomal dominant hypocalcemia (ADH) characterized by low serum calcium, low PTH and relative hypercalciuria. 4 activating CaSR mutations also cause renal wasting of sodium, potassium and other salts (Bartter syndrome type 5). It is not clear why these 4 activating mutations have this additional phenotype. Phosphorylation of CaSR T888 by PKC constitutes an inhibitory feedback mechanism that leads to oscillations of intracellular calcium ([Ca2+]i) at 2 – 4 mM extracellular calcium ([Ca2+]o) but to a more sustained elevation of [Ca2+]i at higher concentrations. We tested for functional differences between wildtype CaSR and mutants causing ADH or Bartter Syndrome Type 5 by investigating oscillatory responses of [Ca2+]i

Impact of Clinically Relevant Mutations on the Pharmacoregulation and Signaling Bias of the Calcium-Sensing Receptor by Positive and Negative Allosteric Modulators

Cinacalcet is predominantly used to treat secondary hyperparathyroidism due to end-stage renal failure, but, more recently, its potential clinical efficacy in treating patients with loss-of-function mutations in the calcium-sensing receptor (CaSR) has been recognized. Many clinically relevant CaSR mutations are located in the heptahelical membrane spanning and extracellular loop regions of the receptor, where allosteric modulators are predicted to bind. The aim of the present study was to investigate the impact of such mutations on the pharmacoregulation of the CaSR by the positive and negative allosteric modulators, cinacalcet and NPS-2143, respectively. Both cinacalcet and NPS-2143 effectively rescued mutants whose cell surface expression was substantially impaired, suggesting that both classes of drug can stabilize a receptor conformation that is trafficked more effectively to the cell surface. In addition, functional impairments in almost all mutant CaSRs were rescued by either cinacalcet or NPS-2143 via restoration of intracellular signaling. There was a significantly greater ability of both compounds to modulate agonist-stimulated intracellular Ca(2+) mobilization than ERK1/2 phosphorylation, indicating that the allosteric modulators engender bias in agonist-stimulated CaSR signaling to different pathways. Three mutations (G(670)R, P(748)R, and L(773)R) altered the binding affinity of allosteric modulators to the CaSR, and 3 mutations (V(817)I, L(773)R, and E(767)K) altered the cooperativity between the allosteric modulator and Ca(2+)(o). These findings have important implications for the treatment of diseases associated with CaSR mutations using allosteric CaSR modulators and for analyzing the effects of mutations on the function and pharmacoregulation of the CaSR.

The intestinal calcistat

The main physiological function of vitamin D is maintenance of calcium homeostasis by its effect on calcium absorption, and bone health in association with parathyroid gland. Vitamin D deficiency (VDD) is defined as serum 25-hydroxy vitamin D (25OHD) levels <20 ng/ml. Do all subjects with VDD have clinical disease according to this definition? We hypothesize that there exist an intestinal calcistat, which controls the calcium absorption independent of PTH levels. It consists of calcium sensing receptor (CaSR) on intestinal brush border, which senses calcium in intestinal cells and vitamin D system in intestinal cells. CaSR dampens the generation of active vitamin D metabolite in intestinal cells and decrease active transcellular calcium transport. It also facilitates passive paracellular diffusion of calcium in intestine. This local adaptation adjusts the fractional calcium absorption according the body requirement. Failure of local adaptation due to decreased calcium intake, decreased supply of 25OHD, mutation in CaSR or vitamin D system decreases systemic calcium levels and systemic adaptations comes into the play. Systemic adaptations consist of rise in PTH and increase in active vitamin D metabolites. These adaptations lead to bone resorption and maintenance of calcium homeostasis. Not all subjects with varying levels of VDD manifest with secondary hyperparathyroidism and decreased in bone mineral density. We suggest that rise in PTH is first indicator of VDD along with decrease in BMD depending on duration of VDD. Hence, subjects with any degree of VDD with normal PTH and BMD should not be labeled as vitamin D deficient. These subjects can be called subclinical VDD, and further studies are required to assess beneficial effect of vitamin D supplementation in this subset of population.

Cinacalcet for the management of hypercalcemia secondary to calcium-sensing receptor mutation

Cinacalcet for the management of hypercalcemia secondary to calcium-sensing receptor mutation

Two novel mutations of the calcium-sensing receptor gene affecting the same amino acid position lead to opposite phenotypes and reveal the importance of p.N802 on receptor activity

Gain-of-function mutations of the calcium-sensing receptor (CASR) gene have been identified in patients with sporadic or familial autosomal dominant hypocalcemia (ADH). Inactivating mutations of the CASR gene cause familial hypocalciuric hypercalcemia (FHH). Here, we report two novel CASR mutations affecting the same amino acid (p.N802); one causes ADH and the other atypical FHH. The first patient, an 11-year-old girl suffering from hypocalcemia, developed nephrocalcinosis when she was only 5 years old. The second patient is a 30-year-old woman who presented with mild hypercalcemia. PCR amplification of CASR coding exons and direct sequencing of PCR products were used to identify mutations. Site-directed mutagenesis was used to generate mutated CASR cDNAs in an expression plasmid. Using the MAPK assay system and transient transfection of Cos-7 cells with wild-type (WT) and mutated CASR, we studied the responses of these mutated receptors to extracellular Ca(2+) and to the negative allosteric CASR modulator, NPS2143. Two heterozygous missense mutations (p.N802I and p.N802S) affecting a residue in the sixth transmembrane domain of CASR were identified. In functional tests, the response of the p.N802S mutant to calcium was typical of an inactivating mutation. However, the p.N802I mutant had 70% of the maximally stimulated WT receptor activity even in the absence of extracellular calcium. This constitutive activity was only partially inhibited by the inhibitor, NPS2143. The asparagine at amino acid position 802 appears to be essential for the activity of the CASR protein and is implicated in the mechanism of CASR signaling.

Minireview: The Intimate Link Between Calcium Sensing Receptor Trafficking and Signaling: Implications for Disorders of Calcium Homeostasis

The calcium-sensing receptor (CaSR) regulates organismal Ca(2+) homeostasis. Dysregulation of CaSR expression or mutations in the CASR gene cause disorders of Ca(2+) homeostasis and contribute to the progression or severity of cancers and cardiovascular disease. This brief review highlights recent findings that define the CaSR life cycle, which controls the cellular abundance of CaSR and CaSR signaling. A novel mechanism, termed agonist-driven insertional signaling (ADIS), contributes to the unique hallmarks of CaSR signaling, including the high degree of cooperativity and the lack of functional desensitization. Agonist-mediated activation of plasma membrane-localized CaSR increases the rate of insertion of CaSR at the plasma membrane without altering the constitutive endocytosis rate, thereby acutely increasing the maximum signaling response. Prolonged CaSR signaling requires a large intracellular ADIS-mobilizable pool of CaSR, which is maintained by signaling-mediated increases in biosynthesis. This model provides a rational framework for characterizing the defects caused by CaSR mutations and the altered functional expression of wild-type CaSR in disease states. Mechanistic dissection of ADIS of CaSR should lead to optimized pharmacological approaches to normalize CaSR signaling in disorders of Ca(2+) homeostasis.

CASR gene activating mutations in two families with autosomal dominant hypocalcemia

BackgroundAutosomal 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. AimTo 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. ResultsWe 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. ConclusionsWe 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.