The role of AP2σ variants in calcitropic and non-calcitropic disorders

The Adaptor Protein 2 (AP2) is a heterotetrameric complex comprising of α, β, μ and σ-subunits, and it has an integral role in clathrin-mediated endocytosis (CME). CME is the process by which plasma membrane constituents are internalised, and this is the major endocytic pathway in mammalian cells. Missense mutations of AP2σ (Arg15Cys, Arg15His and Arg15Leu) cause impaired internalisation of the calcium-sensing receptor (CaSR), a G-protein coupled receptor (GPCR), and lead to Familial Hypocalciuric Hypercalcaemia type 3 (FHH3), a condition associated with moderate to severe hypercalcaemia, which may be symptomatic and associated with low bone mineral density and pancreatitis. Patients with FHH3-associated mutations may also have non-calcitropic disorders, including neurodevelopmental disorders.
This thesis aims to further explore the role of AP2σ variants in calcitropic and non-calcitropic disorders by studying large population datasets to search for novel AP2S1 variants; generating mutant mouse models of FHH3; and investigating the role of AP2σ mutations in neurodevelopmental disorders.
My studies have identified 3 additional AP2S1 variants in humans, Thr112Met, Met117Ile and Glu142Lys, that affect intracellular calcium (Ca2+i) signalling in vitro. Clinical data was available for the Met117Ile only, and the patient had mild hypercalcaemia. The identification of these novel variants potentially increases the spectrum of known mutations in AP2S1, suggesting a change in practice to include exon 5 in addition to exon 2 when screening the gene in FHH patients, and demonstrates the utility of large-scale sequencing databases in identifying genetic variants.
The heterozygote Ap2s1+/L15, Ap2s1+/C15 and Ap2s1+/I117 mice were viable and reached adulthood. However, the homozygote Ap2s1L15/L15 died in the early neonatal period, whilst the Ap2s1C15/C15 mice were subviable with significantly fewer mice surviving weaning and by adulthood, whereas the Ap2s1I117/I117 mice were viable and reached adulthood. The Ap2s1+/L15 and Ap2s1+/C15 mice had hypercalcaemia, with the hypercalcaemia being significantly greater in the Ap2s1+/L15 than the Ap2s1+/C15 mice. The homozygote Ap2s1L15/L15 and Ap2s1C15/C15 mice had significantly greater elevations in plasma calcium than their heterozygote counterparts. The Ap2s1+/I117 and Ap2s1I117/I117 mice did not have hypercalcaemia. In conclusion, my studies have expanded the phenotypic spectrum associated with AP2S1 mutations, and have also established Ap2s1 mouse models that will enable the evaluation of the roles of AP2σ in calcium homeostasis and endosomal trafficking of the CaSR through pathophysiological studies that are difficult to undertake in patients. The Ap2s1+/L15 and the Ap2s1+/C15 mutant mice represent models for human FHH3.
Finally, I describe nine children with heterozygous variants of AP2S1 Arg10 in association with neurodevelopmental delay, thereby suggesting a wider role in non-calcitropic phenotype of AP2S1 mutations.

Keywords: Familial Hypocalciuric Hypercalcaemia type 3
Genetic variants
Neurodevelopmental disorders
Intracellular signalling
Sequencing datasets
Adaptor Protein 2
Advisor: Rorsman, P
Thakker, R
Cox, R
Gorvin, C