Endocrine Abstracts

The calcium-sensing receptor (CaSR) is a guanine-nucleotide-binding protein (G-protein)-coupled receptor that has a central role in calcium homeostasis. Loss-of-function mutations of the CaSR result in familial hypocalciuric hypercalcemia type 1 (FHH1) and gain-of-function mutations in autosomal dominant hypocalcemia (ADH). Recently, loss-of-function Gα₁₁ mutations have been identified to cause FHH2 and we hypothesised that gain-of-function Gα₁₁ mutations may be a cause of ADH in the ≈60% of ADH patients who do not have CaSR mutations. DNA sequence analysis of the 1077 bp coding region and 12 exon-intron boundaries of GNA11 in eight patients with hypocalcaemia but without CaSR mutations identified two missense mutations, Arg181Gln and Phe341Leu. These missense mutations were absent from ≈5400 exomes and both Arg181 and Phe341 residues are highly conserved in vertebrate Gα₁₁ subunit orthologues and human paralogues indicating that these were likely mutations of GNA11. Wild-type (WT) and ADH2-associated mutants Gln181 and Leu341 Gα₁₁ proteins were expressed by transient transfection in HEK293 cells, stably transfected with CaSR, and assessed by measuring their intracellular calcium responses to changes in extracellular calcium ((Ca²⁺)_o). This revealed that expression of the mutant Gα₁₁ proteins resulted in a leftward shift in the concentration-response curves to alterations in (Ca²⁺)_o and a significant (P<0.0001) reduction in EC₅₀ when compared to transfection with WT Gα₁₁ (WT EC₅₀=2.25 mM (95% confidence interval (CI) 2.21–2.29 mM), Gln181 EC₅₀=1.92 mM (95% CI 1.81–2.02 mM), Leu341 EC₅₀=1.99 mM (95% CI 1.83–2.10 mM). This indicates that these mutations are associated with Gα₁₁ gain-of-function. Three-dimensional modelling of these mutations predicted conformational changes resulting in reduced stabilisation of the GTP hydrolysis transition state and a reduction in the intrinsic Gα₁₁ GTPase activity leading to prolonged lifetime of the active GTP-bound Gα₁₁ subunit. Thus, our results establish a new disorder, ADH type 2 (ADH2) which is due to the first reported germline gain-of-function mutations in Gα₁₁.

Declaration of funding

This work was supported by the Medical Research Council programme grants – G9825289 and G1000467 (M A N, F M H, and R V T), and National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme (M A N and R V T); European Commission Seventh Framework Programme (FP7-264663) (V N B); S A H is a Wellcome Trust Clinical Research Training Fellow.