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Identification and functional characterization of loss-of-function mutations of the calcium-sensing receptor in four Italian kindreds with familial hypocalciuric hypercalcemia

¹ , Department of Endocrinology and Metabolism² Unit of General Physiology, Department of Biology, University of Pisa, 56100 Pisa, Italy³ Department of Environmental Sciences, University of Tuscia, Viterbo, Italy⁴ Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA

(Correspondence should be addressed to C Marcocci who is now at Dipartimento di Endocrinologia e Metabolismo, Università di Pisa, Via Paradisa 2, 56124 Pisa, Italy; Email: c.marcocci{at}endoc.med.unipi.it)

Objective: Identification and characterization of calcium-sensing receptor (CASR) mutations in four unrelated Italian kindreds with familial hypocalciuric hypercalcemia.

Design: Clinical evaluation and genetic analysis of CASR gene. Functional characterization of mutated CASRs.

Methods: Direct sequencing of CASR gene in genomic DNA. Studies of CASR-mediated increases in cytosolic calcium concentration [Ca²⁺]_i in CASR-transfected COS-7 cells in vitro.

Results: Four unreported heterozygous CASR mutations 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⁺]_i relative to the wildtype (WT) CASR to increasing extracellular calcium concentrations. Cotransfection experiments showed that the H595Y and P748H mutants did not affect the apparent affinity of the WT CASR for 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 WT CASR to calcium, suggesting that it may reduce the effective concentration of the normal CASR on the cell surface or impair its maximal signaling capacity.

Conclusions: Four CASR mutations were identified. The reduced functional responses to extracellular calcium and normal expression of the mutant receptors suggest that conformational changes account for altered CASR activity. Moreover, a reduced complement of normal CASRs in these heterozygous patients, perhaps combined with a mutant receptor-induced decrease in maximal activity of the WT receptor, may contribute to defective calcium-sensing in vivo.