Endocrine Abstracts (2019) 65 EC1.4 | DOI: 10.1530/endoabs.65.EC1.4

Mice harbouring a germline heterozygous AP2S1 mutation, Arg15Leu, are a model for familial hypocalciuric hypercalcaemia type 3 (FHH3)

Fadil Hannan1, Victoria Stokes1, Caroline Gorvin1, Mark Stevenson1, Tertius Hough2, Michelle Stewart2, Sara Wells2, Lydia Teboul2 & Rajesh Thakker1


1University of Oxford, Oxford, UK; 2MRC Harwell Institute, Oxfordshire, UK


Familial hypocalciuric hypercalcaemia (FHH) comprises three genetic variants: FHH types 1 and 2 are due to mutations of the calcium-sensing receptor (CaSR) and G-protein subunit alpha-11, whereas, FHH type 3 (FHH3) is caused by heterozygous mutations affecting the Arg15 residue (Arg15Cys, Arg15His, Arg15Leu) of the adaptor-related protein complex 2-sigma subunit (AP2S1), which regulates CaSR endocytosis. FHH is usually associated with mild hypercalcaemia, normal parathyroid hormone (PTH) and low urinary calcium excretion. However, FHH3 patients harbouring the Arg15Leu AP2S1 mutation may have marked and symptomatic hypercalcaemia. To further evaluate the impact of the Arg15Leu AP2S1 mutation on calcium homeostasis, we used CRISPR/Cas9-mediated gene editing to generate mice harbouring this mutation (Ap2s1+/L15). Plasma and 24-h urine was collected for biochemical analysis, and bone mineral density (BMD) was measured by DEXA. These mice were also treated with cinacalcet to assess whether this CaSR positive allosteric modulator can rectify any alterations in calcium homeostasis. All studies were conducted in age-matched adult mice and in accordance with institutional welfare guidelines. Male and female Ap2s1+/L15 mice were viable and had marked hypercalcaemia (plasma adjusted-calcium =2.92±0.01 mmol/l) compared to wild-type mice (plasma adjusted-calcium =2.33±0.01 mmol/l, P<0.0001). This finding was associated with significant hypophosphataemia, hypermagnesaemia, increased plasma PTH, and significantly reduced 24-h urine calcium excretion. Furthermore, male Ap2s1+/L15 mice had significantly reduced BMD. Cinacalcet was administered as a 60 mg/kg oral bolus to male and female Ap2s1+/L15 mice, and plasma adjusted-calcium and PTH measured at 0, 1, 2 and 4 h post-dose. Cinacalcet suppressed plasma PTH at 1-h post-dose and maximally lowered plasma-adjusted calcium to 2.51±0.02 mmol/l (P<0.001 compared to the pre-treatment value of 2.93±0.03 mmol/l) at 2-h post-dose. Thus, these studies have established a mouse model for FHH3, and demonstrate that cinacalcet can be used to treat the marked hypercalcaemia and hyperparathyroidism caused by the Arg15Leu AP2S1 mutation.