Dataset Information

Identification and Functional Characterization of a Calcium-Sensing Receptor Mutation in an Infant with Familial Hypocalciuric Hypercalcemia.

ABSTRACT: Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominant disorder, associated with inactivating mutations of the calcium-sensing receptor (CaSR). To evaluate the functional significance of a CaSR mutation, identified in a young infant who presented with hypercalcemia and hypocalciuria. The CaSR gene coding sequences were analyzed by polymerase chain reaction amplification and direct sequencing analysis. The mutation identified was introduced by site-directed mutagenesis into a wild-type (WT) CaSR plasmid, and human embryonic kidney 293 T cells were transfected with either the WT or mutant CaSR. The function of the mutated CaSR protein was analyzed by evaluating the free intracellular calcium [(Ca2+)i] response after challenge with extracellular calcium (Ca2+). We identified a heterozygous mutation c.772_773delGTinsA in exon 4 resulting in the substitution of amino acid valine (Val) with amino acid arginine (Arg) and the premature pause of the translation 46 amino acids later (Val258ArgfsTer47). Functional assay showed that cells transfected with the mutant CaSR had a significantly poorer response to extracellular Ca2+ stimulation compared with the WT. We have shown that the c.772_773delGTinsA mutation causes a significant alteration of CaSR function leading to features of FHH in an affected young infant since the first months of life.

SUBMITTER: Papadopoulou A

PROVIDER: S-EPMC5096500 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Identification and Functional Characterization of a Calcium-Sensing Receptor Mutation in an Infant with Familial Hypocalciuric Hypercalcemia.

Papadopoulou Anna A Gole Evangelia E Melachroinou Katerina K Meristoudis Christos C Siahanidou Tania T Papadimitriou Anastasios A

Journal of clinical research in pediatric endocrinology 20160418 3

Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominant disorder, associated with inactivating mutations of the calcium-sensing receptor (CaSR). To evaluate the functional significance of a CaSR mutation, identified in a young infant who presented with hypercalcemia and hypocalciuria. The CaSR gene coding sequences were analyzed by polymerase chain reaction amplification and direct sequencing analysis. The mutation identified was introduced by site-directed mutagenesis into a wild-ty ...[more]

PMID: 27087013

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Project description:Familial hypocalciuric hypercalcemia (FHH) is a genetically heterogeneous disorder with three variants, FHH1 to FHH3. FHH1 is caused by loss-of-function mutations of the calcium-sensing receptor (CaSR), a G-protein coupled receptor that predominantly signals via G-protein subunit alpha-11 (Gα11 ) to regulate calcium homeostasis. FHH2 is the result of loss-of-function mutations in Gα11 , encoded by GNA11, and to date only two FHH2-associated Gα11 missense mutations (Leu135Gln and Ile200del) have been reported. FHH3 is the result of loss-of-function mutations of the adaptor protein-2 σ-subunit (AP2σ), which plays a pivotal role in clathrin-mediated endocytosis. We describe a 65-year-old woman who had hypercalcemia with normal circulating parathyroid hormone concentrations and hypocalciuria, features consistent with FHH, but she did not have CaSR and AP2σ mutations. Mutational analysis of the GNA11 gene was therefore undertaken, using leucocyte DNA, and this identified a novel heterozygous GNA11 mutation (c.161C>T; p.Thr54Met). The effect of the Gα11 variant was assessed by homology modeling of the related Gαq protein and by measuring the CaSR-mediated intracellular calcium (Ca(2+) i ) responses of HEK293 cells, stably expressing CaSR, to alterations in extracellular calcium (Ca(2+) o ) using flow cytometry. Three-dimensional modeling revealed the Thr54Met mutation to be located at the interface between the Gα11 helical and GTPase domains, and to likely impair GDP binding and interdomain interactions. Expression of wild-type and the mutant Gα11 in HEK293 cells stably expressing CaSR demonstrate that the Ca(2+) i responses after stimulation with Ca(2+) o of the mutant Met54 Gα11 led to a rightward shift of the concentration-response curve with a significantly (p < 0.01) increased mean half-maximal concentration (EC50 ) value of 3.88 mM (95% confidence interval [CI] 3.76-4.01 mM), when compared with the wild-type EC50 of 2.94 mM (95% CI 2.81-3.07 mM) consistent with a loss-of-function. Thus, our studies have identified a third Gα11 mutation (Thr54Met) causing FHH2 and reveal a critical role for the Gα11 interdomain interface in CaSR signaling and Ca(2+) o homeostasis. © 2016 American Society for Bone and Mineral Research.

Project description:G-protein subunit ?-11 (G?11 ) couples the calcium-sensing receptor (CaSR) to phospholipase C (PLC)-mediated intracellular calcium (Ca2+i ) and mitogen-activated protein kinase (MAPK) signaling, which in the parathyroid glands and kidneys regulates parathyroid hormone release and urinary calcium excretion, respectively. Heterozygous germline loss-of-function G?11 mutations cause familial hypocalciuric hypercalcemia type 2 (FHH2), for which effective therapies are currently not available. Here, we report a novel heterozygous G?11 germline mutation, Phe220Ser, which was associated with hypercalcemia in a family with FHH2. Homology modeling showed the wild-type (WT) Phe220 nonpolar residue to form part of a cluster of hydrophobic residues within a highly conserved cleft region of G?11 , which binds to and activates PLC; and predicted that substitution of Phe220 with the mutant Ser220 polar hydrophilic residue would disrupt PLC-mediated signaling. In vitro studies involving transient transfection of WT and mutant G?11 proteins into HEK293 cells, which express the CaSR, showed the mutant Ser220 G?11 protein to impair CaSR-mediated Ca2+i and extracellular signal-regulated kinase 1/2 (ERK) MAPK signaling, consistent with diminished activation of PLC. Furthermore, engineered mutagenesis studies demonstrated that loss of hydrophobicity within the G?11 cleft region also impaired signaling by PLC. The loss-of-function associated with the Ser220 G?11 mutant was rectified by treatment of cells with cinacalcet, which is a CaSR-positive allosteric modulator. Furthermore, in vivo administration of cinacalcet to the proband harboring the Phe220Ser G?11 mutation, normalized serum ionized calcium concentrations. Thus, our studies, which report a novel G?11 germline mutation (Phe220Ser) in a family with FHH2, reveal the importance of the G?11 hydrophobic cleft region for CaSR-mediated activation of PLC, and show that allosteric CaSR modulation can rectify the loss-of-function Phe220Ser mutation and ameliorate the hypercalcemia associated with FHH2. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Dataset Information

Identification and Functional Characterization of a Calcium-Sensing Receptor Mutation in an Infant with Familial Hypocalciuric Hypercalcemia.

Publications

Identification and Functional Characterization of a Calcium-Sensing Receptor Mutation in an Infant with Familial Hypocalciuric Hypercalcemia.

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