MON-539 Mice Harboring a Germline Heterozygous AP2S1 Mutation, Arg15Leu, Are a Model for Familial Hypocalciuric Hypercalcemia Type 3 (FHH3)

Familial hypocalciuric hypercalcemia (FHH) is a disorder of calcium homeostasis comprising three reported genetic variants: FHH types 1 and 2 are due to germline loss-of-function mutations of the calcium-sensing receptor (CaSR) and G-protein subunit α-11 (Gα(11)), respectively, whereas, FHH type 3 (FHH3) is mainly caused by germline heterozygous loss-of-function mutations affecting the Arg15 residue (Arg15Cys, Arg15His or Arg15Leu) of the adaptor-related protein complex 2-sigma subunit (AP2σ), which is encoded by the AP2S1 gene, and regulates CaSR endocytosis and endosomal signaling. FHH is considered to be an asymptomatic disorder characterised by mild-to-moderate hypercalcemia, normal or mildly elevated parathyroid hormone (PTH) concentrations and low urinary calcium excretion. However, some FHH3 patients, especially those harboring the Arg15Leu AP2S1 mutation, have marked and symptomatic hypercalcemia, thereby indicating that FHH3 may represent a more severe clinical disorder. To further evaluate the impact of the Arg15Leu AP2S1 mutation on calcium homeostasis, we utilised CRISPR/Cas9-mediated gene editing to generate C57BL/6J mice harboring the germline Arg15Leu mutation. Plasma and 24-hour urine samples were collected for biochemical analysis, and bone mineral density (BMD) was measured by dual energy X-ray absorptiometry (DEXA). All studies were conducted in mice aged 12-15 weeks (n = 10-12 mice per genotype), and in accordance with institutional welfare guidelines. Wild-type (Ap2s1(+/+)), heterozygous (Ap2s1(+/L15)) and homozygous (Ap2s1(L15/L15)) mice were born as expected for a Mendelian pattern of inheritance, however, Ap2s1(L15/L15) mice died within 48 hours of birth. In contrast, male and female Ap2s1(+/L15)mice were viable and biochemical analysis showed marked hypercalcemia (plasma albumin-adjusted calcium = 2.77±0.02 mmol/L) compared to wild-type (WT) mice (plasma albumin-adjusted calcium = 2.13±0.03 mmol/L, p<0.0001), and this finding was associated with significant hypophosphatemia and hypermagnesemia. Male and female Ap2s1(+/L15)mice also had significant elevations of plasma PTH (157±14 pmol/L) compared to WT mice (63±10 pmol/L, p<0.0001). Furthermore, urine analysis showed that Ap2s1(+/L15)mice were significantly hypocalciuric (fractional excretion of calcium (FECa) = 0.016±0.002) compared to WT mice (FECa =0.029±0.002, p<0.01), whilst, DEXA analysis demonstrated that Ap2s1(+/L15)mice have significantly reduced BMD. Thus, these studies have established a mouse model for FHH3, which highlights the importance of the AP2σ protein in the parathyroid and renal regulation of mineral and bone homeostasis. In addition, these findings demonstrate that the germline heterozygous Arg15Leu AP2S1 mutation causes a severe form of FHH characterized by marked hypercalcemia, hyperparathyroidism and reduced BMD.