A G-protein Subunit-α11 Loss-of-Function Mutation, Thr54Met, Causes Familial Hypocalciuric Hypercalcemia Type 2 (FHH2)

Caroline M Gorvin, Treena Cranston, Fadil M Hannan, Nigel Rust, Asjid Qureshi, M. Andrew Nesbit, Rajesh V Thakker

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

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 (Ca2+i) responses of HEK293 cells, stably expressing CaSR, to alterations in extracellular calcium (Ca2+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 Ca2+i responses after stimulation with Ca2+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 Ca2+o homeostasis. © 2016 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).
LanguageEnglish
Pages1200-1206
JournalJournal of Bone and Mineral Research
Volume31
Issue number6
DOIs
Publication statusPublished - 6 Feb 2016

Fingerprint

Calcium-Sensing Receptors
Protein Subunits
GTP-Binding Proteins
Mutation
Sigma Factor
HEK293 Cells
Calcium
Homeostasis
Confidence Intervals
GTP-Binding Protein alpha Subunits
Clathrin
GTP Phosphohydrolases
Hypercalcemia
Missense Mutation
G-Protein-Coupled Receptors
Endocytosis
Hypocalciuric hypercalcemia, familial, type 2
Parathyroid Hormone
Minerals
Flow Cytometry

Keywords

  • DISORDERS OF CALCIUM/PHOSPHATE METABOLISM
  • PTH/VIT D/FGF23
  • PARATHYROID-RELATED DISORDERS
  • CELL/TISSUE SIGNALING – ENDOCRINE PATHWAYS

Cite this

Gorvin, Caroline M ; Cranston, Treena ; Hannan, Fadil M ; Rust, Nigel ; Qureshi, Asjid ; Nesbit, M. Andrew ; Thakker, Rajesh V. / A G-protein Subunit-α11 Loss-of-Function Mutation, Thr54Met, Causes Familial Hypocalciuric Hypercalcemia Type 2 (FHH2). In: Journal of Bone and Mineral Research. 2016 ; Vol. 31, No. 6. pp. 1200-1206.
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abstract = "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 (Ca2+i) responses of HEK293 cells, stably expressing CaSR, to alterations in extracellular calcium (Ca2+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 Ca2+i responses after stimulation with Ca2+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 Ca2+o homeostasis. {\circledC} 2016 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).",
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A G-protein Subunit-α11 Loss-of-Function Mutation, Thr54Met, Causes Familial Hypocalciuric Hypercalcemia Type 2 (FHH2). / Gorvin, Caroline M; Cranston, Treena; Hannan, Fadil M; Rust, Nigel; Qureshi, Asjid; Nesbit, M. Andrew; Thakker, Rajesh V.

In: Journal of Bone and Mineral Research, Vol. 31, No. 6, 06.02.2016, p. 1200-1206.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A G-protein Subunit-α11 Loss-of-Function Mutation, Thr54Met, Causes Familial Hypocalciuric Hypercalcemia Type 2 (FHH2)

AU - Gorvin, Caroline M

AU - Cranston, Treena

AU - Hannan, Fadil M

AU - Rust, Nigel

AU - Qureshi, Asjid

AU - Nesbit, M. Andrew

AU - Thakker, Rajesh V

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N2 - 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 (Ca2+i) responses of HEK293 cells, stably expressing CaSR, to alterations in extracellular calcium (Ca2+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 Ca2+i responses after stimulation with Ca2+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 Ca2+o homeostasis. © 2016 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).

AB - 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 (Ca2+i) responses of HEK293 cells, stably expressing CaSR, to alterations in extracellular calcium (Ca2+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 Ca2+i responses after stimulation with Ca2+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 Ca2+o homeostasis. © 2016 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).

KW - DISORDERS OF CALCIUM/PHOSPHATE METABOLISM

KW - PTH/VIT D/FGF23

KW - PARATHYROID-RELATED DISORDERS

KW - CELL/TISSUE SIGNALING – ENDOCRINE PATHWAYS

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DO - 10.1002/jbmr.2778

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JO - Journal of Bone and Mineral Research

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JF - Journal of Bone and Mineral Research

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ER -