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

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).

Knockin mouse with mutant Gα11 mimics human inherited hypocalcemia and is rescued by pharmacologic inhibitors

“…The peak mean fluorescence ratio of the Ca 2+ i transient response after each individual stimulus was measured using Cytomation Summit software (Beckman Coulter) and expressed as a normalized response, as described (11,35). Nonlinear regression of concentration-response curves was performed with GraphPad Prism using the normalized response at each [Ca 2+ ] o for each separate experiment for the determination of EC 50 (i.e., [Ca 2+ ] o required for 50% of the maximal response) and Hill coefficient values (36). The maximal signaling response was measured as a fold-change of the peak transient Ca ] o (36).…”

“…Nonlinear regression of concentration-response curves was performed with GraphPad Prism using the normalized response at each [Ca 2+ ] o for each separate experiment for the determination of EC 50 (i.e., [Ca 2+ ] o required for 50% of the maximal response) and Hill coefficient values (36). The maximal signaling response was measured as a fold-change of the peak transient Ca ] o (36). The maximal signaling responses for mutant Gα 11 proteins were expressed as a percentage of the WT Gα 11 protein maximal signaling response.…”

“…The maximal signaling responses for mutant Gα 11 proteins were expressed as a percentage of the WT Gα 11 protein maximal signaling response. The mean EC 50 and Hill coefficients obtained from 4 separate transfection experiments were used for statistical comparison by using the F-test, and alterations in maximal signaling responses assessed using the Mann-Whitney U test (36). signaling.…”

The Calcium-Sensing Receptor and the Parathyroid: Past, Present, Future

“…These findings demonstrate that G q and G 11 are required for CaSR-mediated control of PTH secretion and thus lie at the top of a key inhibitory signaling pathway(s). Consistent with these findings, inactivating and activating mutations of the human Gα 11 gene have been shown respectively to underlie variant forms of FHH (FHH2) and ADH (ADH2; Nesbit et al, 2013a; Gorvin et al, 2016; Piret et al, 2016). …”

Genetics of Hyperparathyroidism, Including Parathyroid Cancer

“…Type 2 FHH (FHH2) due to germline loss-of-function mutation of GNA11 , encoding the G protein α11 subunit 61,62 , and type 3 FHH (FHH3) due to germline loss-of-function mutation in AP2S1 , encoding the adaptor protein-2 sigma subunit involved in clathrin-mediated endocytosis, 63–66 have been recently described. Somatic mutation of neither GNA11 nor AP2S1 has been reported in sporadic parathyroid tumors.…”