Abstract
Adaptor protein 2 (AP2), a heterotetrameric complex comprising AP2α, AP2β2, AP2μ2 and AP2σ2 subunits, is ubiquitously expressed and involved in endocytosis and trafficking of membrane proteins, such as the calcium-sensing receptor (CaSR), a G-protein coupled receptor that signals via Gα11. Mutations of CaSR, Gα11 and AP2σ2, encoded by AP2S1, cause familial hypocalciuric hypercalcaemia types 1–3 (FHH1–3), respectively. FHH3 patients have heterozygous AP2S1 missense Arg15 mutations (p.Arg15Cys, p.Arg15His or p.Arg15Leu) with hypercalcaemia, which may be marked and symptomatic, and occasional hypophosphataemia and osteomalacia. To further characterize the phenotypic spectrum and calcitropic pathophysiology of FHH3, we used CRISPR/Cas9 genome editing to generate mice harboring the AP2S1 p.Arg15Leu mutation, which causes the most severe FHH3 phenotype. Heterozygous (Ap2s1+/L15) mice were viable, and had marked hypercalcaemia, hypermagnesaemia, hypophosphataemia, and increases in alkaline phosphatase activity and fibroblast growth factor-23. Plasma 1,25-dihydroxyvitamin D was normal, and no alterations in bone mineral density or bone turnover were noted. Homozygous (Ap2s1L15/L15) mice invariably died perinatally. Co-immunoprecipitation studies showed that the AP2S1 p.Arg15Leu mutation impaired protein–protein interactions between AP2σ2 and the other AP2 subunits, and also with the CaSR. Cinacalcet, a CaSR positive allosteric modulator, decreased plasma calcium and parathyroid hormone concentrations in Ap2s1+/L15 mice, but had no effect on the diminished AP2σ2-CaSR interaction in vitro. Thus, our studies have established a mouse model that is representative for FHH3 in humans, and demonstrated that the AP2S1 p.Arg15Leu mutation causes a predominantly calcitropic phenotype, which can be ameliorated by treatment with cinacalcet.
Highlights
Familial hypocalciuric hypercalcaemia (FHH) is an autosomal dominant disorder of extracellular calcium metabolism characterized by lifelong increases of serum calcium concentrations, mild hypermagnesaemia, normal or elevated circulating parathyroid hormone (PTH) concentrations, and inappropriately low urinary calcium excretion (urine calcium to creatinine clearance ratio (CCCR) < 0.01) [1]
We have established by the use of CRISPR/Cas9 genome editing, a mouse model for FHH3, and this will enable the calcitropic roles of AP2σ 2 and endosomal trafficking of the calcium-sensing receptor (CaSR) to be further evaluated together with pursuit of pathophysiological studies that are difficult to undertake in patients with this condition
Our results revealed that Ap2s1+/L15 mice, which harbored a germline heterozygous Ap2s1 p.Arg15Leu mutation, had a similar plasma biochemical phenotype to that reported for FHH3 patients, who have heterozygous loss-of-function AP2S1 missense Arg15 mutations (p.Arg15Cys, p.Arg15His or p.Arg15Leu), and with those having the AP2σ 2 p.Arg15Leu mutation being affected with the severest hypercalcaemia [3,7]
Summary
Familial hypocalciuric hypercalcaemia (FHH) is an autosomal dominant disorder of extracellular calcium metabolism characterized by lifelong increases of serum calcium concentrations, mild hypermagnesaemia, normal or elevated circulating parathyroid hormone (PTH) concentrations, and inappropriately low urinary calcium excretion (urine calcium to creatinine clearance ratio (CCCR) < 0.01) [1]. FHH3 is caused by germline heterozygous lossof-function mutations of the AP2S1 gene, which is located on chromosome 19q13.3 and encodes the AP2σ 2 protein [7]. AP2S1 mutations have been reported in ∼70 FHH probands to-date, and affected individuals harbor a mutation affecting the AP2σ 2 Arg residue, which may give rise to a p.Arg15Cys, p.Arg15His or p.Arg15Leu missense mutation [3,5,6,7,8,9,10,11]. FHH3 patients harboring the p.Arg15Leu AP2σ 2 mutation have been reported to have greater hypercalcaemia and to present at an earlier age than probands with p.Arg15Cys or p.Arg15His AP2σ 2 mutations [3]
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