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

Abstract

Objective: 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. Patients and methods: 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 2C and to the negative allosteric CASR modulator, NPS2143. Results: 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. Conclusions: 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.

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

“…Only two of 4 known CaSR mutants were sensitive to NPS-2143 either expressed alone or when co-expressed with wt-CaSR to approximate heterozygosity in patients. This resembles previous data from us and others showing that some but not all ADH CaSR mutants were sensitive to NPS-2143 [20] , [34] , [35] . Thus, amino alcohol derived calcilytics may not be the ideal drugs to treat disorders caused by activating CaSR mutations.…”

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)

“…NPS 2143 was used as it induces more prolonged elevations in circulating PTH concentrations than other calcilytic drugs ( 17 , 31 ) and has been shown to be safe and well tolerated in rodent studies ( 15 ). However, previous in vitro studies have indicated that NPS 2143 may not be a suitable therapy for all gain-of-function CaSR mutations ( 19 – 22 , 25 ). Indeed, ADH-associated CaSR mutations, such as Ala835Asp and Ala843Glu, which are located close to the Glu837 TMD residue that is critical for NPS 2143 binding (Supplemental Figure 1) ( 19 , 21 , 32 ), have been demonstrated to completely abrogate the action of NPS 2143, whereas ADH-associated TMD CaSR mutations located away from known NPS 2143 binding residues, such as Glu767Gln, Leu773Arg and Asn802Ile, may partially diminish the efficacy of this calcilytic compound (Supplemental Figure 1) ( 20 , 22 , 25 ).…”

“…Moreover, prolonged exposure of cells expressing gain-of-function mutant CaSRs to calcilytic drugs may facilitate internalization of these allosteric modulators, which then bind and destabilize the conformation of nascent CaSRs, thus leading to enhanced proteasomal degradation of mutant receptors ( Figure 1 ) ( 7 , 8 ). However, some gain-of-function mutations located within the CaSR transmembrane domain (TMD), which is predicted to be the binding site for allosteric CaSR modulators ( 23 , 24 ), have been shown to impair the effectiveness of calcilytic drugs ( Supplemental Figure 1 ) ( 19 – 22 , 25 ). Moreover, it is unclear whether calcilytic drugs may improve the hypocalcemia of ADH1 while minimizing the risk of hypercalciuric renal disease.…”

“…Nuf mice were originally identified for having opaque flecks in the nucleus of the lens and have an ADH phenotype characterized by hypocalcemia, hyperphosphatemia, inappropriately reduced plasma PTH concentrations, and ectopic calcification ( 26 ). Before embarking on an in vivo assessment of the efficacy of the calcilytic NPS 2143 in rectifying the Nuf mouse hypocalcemia, we used first an in vitro functional assay to determine whether NPS 2143 may normalize the gain-of-function associated with the Nuf Leu723Gln mutation, because some TMD-located ADH-associated CaSR mutations have been reported to be unresponsive or partially responsive to calcilytic drugs ( 19 – 22 , 25 ).…”

GENETICS IN ENDOCRINOLOGY: Gain and loss of function mutations of the calcium-sensing receptor and associated proteins: current treatment concepts

“…NPS-2143 and its close relative ronacaleret are amino-alcohols, whereas ATF936 and AXT914 are quinazolinones. These different classes of calcilytics have partly different bindings sites (48) and data from four publications show that some but not all ADH CASR mutants were sensitive to NPS-2143 (108,111,112,113). In contrast all five ADH and all four BS5 mutants tested so far were sensitive to ATF936 and AXT914 (111).…”