ObjectiveIdentification and characterization of calcium-sensing receptor (CASR) mutations in four unrelated Italian kindreds with familial hypocalciuric hypercalcemia.DesignClinical evaluation and genetic analysis ofCASRgene. Functional characterization of mutatedCASRs.MethodsDirect sequencing ofCASRgene in genomic DNA. Studies ofCASR-mediated increases in cytosolic calcium concentration [Ca2+]iinCASR-transfected COS-7 cellsin vitro.ResultsFour unreported heterozygousCASRmutations were identified, including three missense (H595Y, P748H, and C765W) and one splice site (IVS2+1G>C) mutation. The H595Y, P748H, and C765W mutant receptors, although expressed at normal levels on the cell surface, showed a reduced response in [Ca2+]irelative to the wildtype (WT)CASRto increasing extracellular calcium concentrations. Cotransfection experiments showed that the H595Y and P748H mutants did not affect the apparent affinity of the WTCASRfor calcium, suggesting that they do not exert a dominant-negative effect. On the other hand, the co-transfected C765W mutant decreased the maximum response of the WTCASRto calcium, suggesting that it may reduce the effective concentration of the normalCASRon the cell surface or impair its maximal signaling capacity.ConclusionsFourCASRmutations were identified. The reduced functional responses to extracellular calcium and normal expression of the mutant receptors suggest that conformational changes account for alteredCASRactivity. Moreover, a reduced complement of normalCASRsin these heterozygous patients, perhaps combined with a mutant receptor-induced decrease in maximal activity of the WT receptor, may contribute to defective calcium-sensingin vivo.
Identification and Functional Characterization of Novel Calcium-Sensing Receptor Mutations in Familial Hypocalciuric Hypercalcemia and Autosomal Dominant Hypocalcemia