SFEBES2013 Oral Communications Obesity, metabolism and bone (8 abstracts)
Familial hypocalciuric hypercalcaemia type 3 is caused by mutations in adaptor protein 2 sigma 1
M Andrew Nesbit 1 , Fadil M Hannan 1 , Sarah A Howles 1 , Anita A C Reed 1 , Treena Cranston 2 , Clare E Thakker 1 , Lorna Gregory 3 , Andrew J. Rimmer 3 , Nigel Rust 4 , Una Graham 5 , Patrick J Morrison 6 , Steven J Hunter 5 , Michael P Whyte 7 & Rajesh V Thakker 1
1Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; 2Oxford Medical Genetics Laboratories, Oxford University Hospitals NHS Trust, Oxford, UK; 3Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK; 4Sir William Dunn School of Pathology, University of Oxford, Oxford, UK; 5Regional Centre for Endocrinology and Diabetes, Royal Victoria Hospital, Belfast, UK; 6Department of Medical Genetics, Queen’s University Belfast, Belfast City Hospital, Belfast, UK; 7Shriners Hospital for Children, Saint Louis, USA.
Familial hypocalciuric hypercalcaemia (FHH) is an autosomal dominant disorder characterized by lifelong elevation of serum calcium concentrations with inappropriately low urinary calcium excretion. Three types referred to as FHH1, FHH2 and FHH3 and located on chromosomes 3q21.1, 19p and 19q13.3, respectively, have been reported. FHH1, caused by loss-of-function mutations of the calcium-sensing receptor (CaSR), accounts for >65% of FHH patients. To identify the genetic defect in FHH3, we performed exome sequencing in patients from two unrelated FHH3 kindreds. This revealed a C to T transition, that predicted occurrence of an Arg15Cys missense mutation, in adaptor protein 2 sigma 1 (AP2S1), encoding AP2σ2, which was demonstrated to co-segregate with FHH3 in 32 affected members from five generations of the two kindreds. This mutation is predicted to alter an evolutionary conserved arginine residue. To determine the frequency of AP2S1 mutations in the ∼35% of FHH patients without CaSR mutations, we undertook DNA sequence analysis of AP2S1 in 50 additional unrelated patients. This revealed occurrence of 11 missense heterozygous mutations, consistent with autosomal dominant inheritance of FHH3, that all affected Arg15 and consisted of four Arg15Cys, three Arg15His, and four Arg15Leu mutations. Wild-type and mutant AP2σ2 were transiently expressed in HEK293 cells, stably transfected with CaSR, and assessed for their response to changes in extracellular calcium levels. This demonstrated that mutant AP2σ2 decreased the sensitivity of these cells to extracellular calcium and reduced CaSR endocytosis. AP2σ2 forms part of the AP2 complex that has a role in G protein-coupled receptor recycling, and examination of the crystal structure of AP2 revealed that replacement of the Arg15 residue of AP2σ2 compromises a key contact with acidic dileucine motifs of cargo proteins. Thus, our studies have identified the genetic defect underlying FHH3, and give important insights into calcium homeostasis.
Declaration of funding
This work was supported by the Medical Research Council (MRC) (Grants numbers G9825289 and G1000467, G0900747/91070), National Institute for Health Research Oxford Biomedical Research Centre Programme, Wellcome Trust (Grant reference 090532/Z/09/Z), Research and Development Office, Northern Ireland, and the Shriners Hospitals for Children (Grant number 15958).
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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