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Abdallah Al-Salameh, Filomena Cetani, Elena Pardi, Carmen Vulpoi, Peggy Pierre, Loïc de Calan, Serge Guyetant, Xavier Jeunemaitre and Pierre Lecomte


The calcium-sensing receptor (CASR) has an important role in calcium homoeostasis by controlling PTH secretion and renal calcium handling. Inactivating mutations in the CASR gene (HGNC ID: 1514) cause familial hypocalciuric hypercalcaemia (FHH). We present a case of FHH patient to describe a novel mutation in the CASR.

Subjects and methods

A 34-year-old patient was referred because of recurrent hypercalcaemia after resection of two hyperplastic parathyroids. Extensive evaluation found elevated PTH and low calcium/creatinine clearance ratio. One of her three children had high serum calcium concentrations. Genetic studies were performed by PCR amplification of CASR coding exons and direct sequencing of PCR products. Transient transfection of the wild-type (WT) CASR and the mutant CASR into COS-7 was performed to assess functional impact of the mutation and the capacity of either protein to mediate increases in cellular levels of inositol phosphates (IPs).


CASR sequencing found a previously undescribed heterozygous base substitution, determining a change of threonine to isoleucine at codon 550 (p.T550I) in the sixth exon. In contrast to those transfected with WT CASR, which showed a five- to eightfold increase in total IPs at high levels of calcium, COS-7 cells transfected with the (p.T550I) mutant showed no increase confirming to the inactivating nature of the mutation. COS-7 cells co-transfected with the WT and the (p.T550I) mutant showed an intermediate response suggesting a possible dominant negative effect.


This case report presents a not-yet-described mutation in the cysteine-rich region of the CASR extracellular domain, a mutation with a possible dominant negative effect.

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Filomena Cetani, Elena Ambrogini, Paolo Viacava, Elena Pardi, Giovanni Fanelli, Antonio Giuseppe Naccarato, Simona Borsari, Monica Lemmi, Piero Berti, Paolo Miccoli, Aldo Pinchera and Claudio Marcocci

Objective: HRPT2 gene mutations are associated with parathyroid carcinomas, and absence of parafibromin immunoreactivity has been suggested as a diagnostic marker of malignancy. The aim of our study was to extend parafibromin studies in a series of benign and malignant parathyroid tumors and cross-validate the results of immunohistochemistry with those of HRPT2 analysis.

Design and patients: We performed parafibromin and cyclin D1 immunostaining and HRPT2 gene analysis using loss of heterozygosity studies and sequencing analysis in parathyroid specimens from 11 patients with carcinoma (eleven primary tumors, one skin, and four lung metastases), 22 with sporadic adenomas, and 4 with atypical adenomas.

Results: Ten out of eleven parathyroid cancers were negative for parafibromin staining and showed HRPT2 gene abnormalities. The remaining sample was negative for immunostaining and genetic analyses. All but one sporadic adenomas showed parafibromin immunoreactivity and no HRPT2 gene abnormalities. The sample with negative immunostaining carried an HRPT2 mutation. Two atypical adenomas were positive and two negative with parafibromin staining. No HRPT2 abnormalities were found in these samples. Cyclin D1 expression was heterogeneous and there was no relationship between expression/expression level of cyclin D1 and parafibromin expression.

Conclusions: We have shown that negative parafibromin staining is almost invariably associated with HRPT2 mutations and confirm that loss of parafibromin staining strongly predicts parathyroid malignancy. In clinical practice, these tests could be particularly useful in the subset of parathyroid tumors with equivocal histological examination. However, their diagnostic value in this setting remains to be proven.

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Filomena Cetani, Monica Lemmi, Davide Cervia, Simona Borsari, Luisella Cianferotti, Elena Pardi, Elena Ambrogini, Chiara Banti, Edward M Brown, Paola Bagnoli, Aldo Pinchera and Claudio Marcocci


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


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


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.


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.


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.