An Adaptor Protein 2 Sigma Subunit (AP2σ) Met117Ile Mutation within a Helix-Loop-Helix Structure Causes Familial Hypocalciuric Hypercalcaemia Type-3

Presentation Number: SAT 357
Date of Presentation: April 1st, 2017

Caroline M Gorvin*1, Raghu Metpally2, John D Overton3, Jeffrey G Reid3, Gerda E Breitwieser4 and Rajesh V Thakker1
1University of Oxford, Oxford, United Kingdom, 2Geisinger Clinic, Weis Center for Research, Danville, PA, 3Regeneron Genetics Center, Tarrytown, NY, 4Weis Center for Research, Danville, PA


Mutations of the sigma subunit of the clathrin-mediated endocytic adaptor protein-2 (AP2σ), encoded by the AP2S1 gene, impair internalisation of the calcium-sensing receptor (CaSR), a G-protein coupled receptor (GPCR), and cause familial hypocalciuric hypercalcemia type-3 (FHH3). To date, AP2σ mutations have only been identified at one residue, Arg15 (R15). We hypothesized that additional rare AP2σ variants, identified in large-scale sequencing projects may be associated with altered in vitro activity, and associated with hypercalcemia in humans. Using the Geisinger Health System-Regeneron DiscovEHR exome sequencing dataset on 51,289 individuals, we identified four patients with heterozygous AP2S1 variants and hypercalcaemia (i.e. plasma calcium concentrations (p[Ca] of >10.0mg/dL). Two patients had the previously reported Arg15His (R15H) mutation, with p[Ca] of 11.4 and 10.5 mg/dL, and two other patients, each of whom had a p[Ca] of 10.1 mg/dL, had novel heterozygous AP2σ variants, Phe52Tyr (F52Y) and Met117Ile (M117I). F52 and M117 are conserved residues in >175,000 human sequences from the NHLBI exome sequencing project and the Exome Aggregation Consortium, and in vertebrate AP2σ subunit orthologues, thereby indicating that the substitutions Y52 and I117 likely represent AP2σ mutations rather than polymorphic variants. Three-dimensional modelling predicted that F52Y, which is located within AP2σ β3, would likely have no effect on the structure of AP2σ, but that the M117I, which is within a helix-loop-helix structure that provides structural stability to the AP2 complex, would reduce the flexibility of AP2σ and hinder AP2 complex movement that is required for its activation. To further assess the functional consequences of these variants we expressed AP2σ wild-type and mutant proteins in HEK293 cells stably expressing CaSR, and measured intracellular calcium responses to elevations in extracellular calcium using flow cytometry and the calcium binding dye Indo-1. F52Y had a mean half-maximal concentration (EC50) value that was not significantly different to the wild-type AP2σ, whereas M117I led to a rightward shift of the dose-response curve with a significantly increased EC50, consistent with a loss-of-function (Wild-type EC50 = 3.47mM (95% confidence interval (CI) = 3.32-3.62mM), F52Y EC50 = 2.70mM (95%CI = 2.70-2.95mM), and M117I EC50 = 2.89mM (95%CI = 2.76-3.00mM)). Furthermore, treatment of cells expressing AP2σ-M117I with the CaSR positive allosteric modulator cinacalcet reduced the EC50 to 2.59mM (95%CI = 2.40-2.80mM), such that it was not significantly different to wild-type. Thus, our studies have identified that mutations in residues other than R15 can lead to AP2σ loss-of-function, and indicate an important role for the AP2σ helix-loop-helix structure.


Disclosure: JDO: Management Position, Regeneron Pharmaceuticals, Inc.. JGR: Management Position, Regeneron Pharmaceuticals, Inc.. Nothing to Disclose: CMG, RM, GEB, RVT