Identification and functional analysis of a novel CaSR mutation in a family with familial hypocalciuric hypercalcemia.

The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that plays an essential role in maintaining calcium homeostasis. In the present study, we analyzed the CaSR gene in a Korean family with familial hypocalciuric hypercalcemia (FHH). Genetic studies were performed by direct sequence analysis of the CaSR gene in genomic DNA obtained from peripheral leukocytes. A novel heterozygous G to T substitution at nucleotide position 1711 in exon 6, resulting in the G571W mutation, was identified in the CaSR gene in a 26-year-old female with asymptomatic hypercalcemia, a low calcium/creatinine clearance ratio, and normal intact parathyroid hormone. To study CaSR expression, the mutation was introduced by site-directed mutagenesis into a wild-type (WT) CaSR-expressing pCR3.1 vector, and COS-7 cells were transfected with either the WT or mutant CaSR-containing vector. Transfected cells loaded with Fura-2/AM, a fluorescent indicator of Ca2+, were assessed for CaSR function by the change in intracellular calcium [as measured by the 340 nm/380 nm fluorescence intensity ratio (F340/F380)] made in response to challenge with extracellular Ca2+. Both WT and G571W cells had equivalent amounts of CaSR protein in the cell membrane. However, after challenge with extracellular Ca2+, cells transfected with G571W CaSR responded with a lower F340/F380 ratio than those transfected with WT CaSR and showed decreased sensitivity to extracellular Ca2+ concentrations. The G571W mutation had therefore impaired the CaSR function. In conclusion, we identified a novel loss-of-function mutation, G571W, in the CaSR gene in a Korean family with FHH.