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

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 Ca2+ 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. European Journal of Endocrinology 168 K27-K34