title: Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype-phenotype correlations, codon bias and dominant-negative effects.
abstract: The adaptor protein-2 sigma subunit (AP2σ2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2σ2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca(2+) o) homeostasis. To elucidate the role of AP2σ2 in Ca(2+) o regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2σ2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2σ2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype-phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2σ2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2σ2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2σ2 mutations to result in a more severe FHH phenotype with genotype-phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.
authors.affiliation: Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
authors.forename: Fadil M
authors.initials: FM
authors.lastname: Hannan
authors.affiliation: Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Howles
authors.affiliation: Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
authors.forename: Angela
authors.initials: A
authors.lastname: Rogers
authors.affiliation: Oxford Molecular Genetics Laboratory, Churchill Hospital, Oxford, UK.
authors.forename: Treena
authors.initials: T
authors.lastname: Cranston
authors.affiliation: Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
authors.forename: Caroline M
authors.initials: CM
authors.lastname: Gorvin
authors.affiliation: Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
authors.forename: Valerie N
authors.initials: VN
authors.lastname: Babinsky
authors.affiliation: Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
authors.forename: Anita A
authors.initials: AA
authors.lastname: Reed
authors.affiliation: Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
authors.forename: Clare E
authors.initials: CE
authors.lastname: Thakker
authors.affiliation: Renal Unit, Great Ormond Street Hospital for Children NHS Foundation Trust and UCL Institute of Child Health, London, UK.
authors.forename: Detlef
authors.initials: D
authors.lastname: Bockenhauer
authors.affiliation: Division of Endocrinology, Boston Children's Hospital, Boston, MA, USA.
authors.forename: Rosalind S
authors.initials: RS
authors.lastname: Brown
authors.affiliation: School of Medicine, Ninewells Hospital, University of Dundee, Dundee, UK.
authors.forename: John M
authors.initials: JM
authors.lastname: Connell
authors.affiliation: Clinical Genetics Department, Sheffield Children's Hospital NHS Foundation Trust, Sheffield, UK.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Cook
authors.affiliation: Queen Elizabeth II Hospital, Welwyn Garden City, UK.
authors.forename: Ken
authors.initials: K
authors.lastname: Darzy
authors.affiliation: Department of Paediatric Endocrinology, Royal Manchester Children's Hospital, Manchester, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: Ehtisham
authors.affiliation: Regional Centre for Endocrinology and Diabetes, Royal Victoria Hospital, Belfast, UK.
authors.forename: Una
authors.initials: U
authors.lastname: Graham
authors.affiliation: Department of Paediatrics, Evelina London Children's Hospital, St. Thomas' Hospital, London, UK.
authors.forename: Tony
authors.initials: T
authors.lastname: Hulse
authors.affiliation: Regional Centre for Endocrinology and Diabetes, Royal Victoria Hospital, Belfast, UK.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Hunter
authors.affiliation: Department of Clinical Genetics, Guy's Hospital, London, UK.
authors.forename: Louise
authors.initials: L
authors.lastname: Izatt
authors.affiliation: Institute of Cancer and Genetics, University Hospital of Wales, Cardiff, UK.
authors.forename: Dhavendra
authors.initials: D
authors.lastname: Kumar
authors.affiliation: Department of Endocrinology, St. Vincent's University Hospital, Dublin, Ireland.
authors.forename: Malachi J
authors.initials: MJ
authors.lastname: McKenna
authors.affiliation: Metabolic Unit, Western General Hospital, NHS Lothian and University of Edinburgh, Edinburgh, UK.
authors.forename: John A
authors.initials: JA
authors.lastname: McKnight
authors.affiliation: Centre for Cancer Research and Cell Biology, Queens University of Belfast, Belfast, UK, Department of Genetic Medicine, Belfast HSC Trust, Belfast, UK.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Morrison
authors.affiliation: Department of Paediatric Endocrinology, Royal Manchester Children's Hospital, Manchester, UK.
authors.forename: M Zulf
authors.initials: MZ
authors.lastname: Mughal
authors.affiliation: Department of Endocrinology, Cork University Hospital, Cork, Ireland.
authors.forename: Domhnall
authors.initials: D
authors.lastname: O'Halloran
authors.affiliation: Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK.
authors.forename: Simon H
authors.initials: SH
authors.lastname: Pearce
authors.affiliation: SE Scotland Genetic Service, Western General Hospital, Edinburgh, UK.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Porteous
authors.affiliation: Department of Endocrinology, Northwick Park Hospital, London, UK.
authors.forename: Mushtaqur
authors.initials: M
authors.lastname: Rahman
authors.affiliation: Diabetes and Endocrine Centre, Royal Bournemouth Hospital, Bournemouth, UK.
authors.forename: Tristan
authors.initials: T
authors.lastname: Richardson
authors.affiliation: Department of Endocrinology, Chesterfield Royal Hospital NHS Foundation Trust, Derbyshire, UK.
authors.forename: Robert
authors.initials: R
authors.lastname: Robinson
authors.affiliation: Pediatric Gastroenterology, Hepatology and Nutrition Unit, Cliniques Universitaires Saint-Luc, Brussels, Belgium.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Scheers
authors.affiliation: Department of Endocrinology, Russells Hall Hospital, Dudley, UK.
authors.forename: Haroon
authors.initials: H
authors.lastname: Siddique
authors.affiliation: Renal Unit, Great Ormond Street Hospital for Children NHS Foundation Trust and UCL Institute of Child Health, London, UK.
authors.forename: William G
authors.initials: WG
authors.lastname: Van't Hoff
authors.affiliation: Department of Clinical Biochemistry, Frimley Park Hospital, Surrey, UK and.
authors.forename: Timothy
authors.initials: T
authors.lastname: Wang
authors.affiliation: Center for Metabolic Bone Disease and Molecular Research, Shriners Hospital for Children, St. Louis, Missouri, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Whyte
authors.affiliation: Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
authors.forename: M Andrew
authors.initials: MA
authors.lastname: Nesbit
authors.affiliation: Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford, UK, rajesh.thakker@ndm.ox.ac.uk.
authors.forename: Rajesh V
authors.initials: RV
authors.lastname: Thakker
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000737:chemistry / Q000235:genetics*; D034002:Adaptor Protein Complex sigma Subunits / Q000737:chemistry / Q000235:genetics*; D000293:Adolescent; D000328:Adult; D019943:Amino Acid Substitution; D015415:Biomarkers; D002460:Cell Line; D002648:Child; D002675:Child, Preschool; D003062:Codon; D003937:Diagnosis, Differential; D005260:Female; D015870:Gene Expression; D005799:Genes, Dominant; D056726:Genetic Association Studies; D006801:Humans; D006934:Hypercalcemia / Q000151:congenital* / Q000175:diagnosis / Q000235:genetics; D007223:Infant; D008297:Male; D008875:Middle Aged; D008958:Models, Molecular; D009154:Mutation; D010375:Pedigree; D010641:Phenotype; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D055815:Young Adult
chemical_list: C510065:AP2S1 protein, human; D033962:Adaptor Protein Complex 2; D034002:Adaptor Protein Complex sigma Subunits; D015415:Biomarkers; D003062:Codon