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Endocrine Abstracts (2013) 31 OC1.7 | DOI: 10.1530/endoabs.31.OC1.7

SFEBES2013 Oral Communications Young Endocrinologists prize session (8 abstracts)

Autosomal dominant hypocalcemia type 2 is caused by germline GNA11 gain-of-function mutations

Sarah Howles 1 , Andrew Nesbit 1 , Fadil Hannan 1 , Valerie Babinsky 1 , Rosie Head 1 , Treena Cranston 2 , Nigel Rust 3 & Rajesh Thakker 1

1Academic Endocrine Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; 2Oxford Molecular Genetics Laboratory, Churchill Hospital, Oxford, UK; 3Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

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.

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