title: Calcium-sensing-related gene mutations in hypercalcaemic hypocalciuric patients as differential diagnosis from primary hyperparathyroidism: detection of two novel inactivating mutations in an Italian population.
abstract: BACKGROUND
  Inactivating mutations of the calcium-sensing receptor (CaSR), of the G-protein subunit α11 (GNA11) and of the adaptor-related protein complex 2, sigma 1 subunit (AP2S1) genes are responsible for familial hypocalciuric hypercalcaemia (FHH). The aim of this study was to analyse prevalence and pathogenicity of CaSR, GNA11 and AP2S1 mutations in patients with an FHH phenotype and to compare them with a sample of patients with primary hyperparathyroidism (PHPT) in order to identify the most useful laboratory parameter for a differential diagnosis.
  
  
  METHODS
  Patients with an FHH phenotype were studied with polymerase chain reaction amplification and direct sequencing of the entire CaSR, GNA11 and AP2S1 coding sequences. Novel mutations were introduced in a Myc-tagged human wild-type (WT) CaSR cDNA-expressing vector, and functional assay was performed on human embryonic kidney cells evaluating expression and function of mutated proteins.
  
  
  RESULTS
  Among 16 FHH patients, none had an inactivating GNA11 or AP2S1 mutation while 3 (18.8%) carried a CaSR mutation and 10 (62.5%) at least one CaSR polymorphism. Within the latter group, 7 of 10 patients had more than one polymorphism (4.1 ± 2.1 per patient). Two novel CaSR mutations [c.2120A>T (E707V) and c.2320G>A (G774S)] were identified: the E707V mutation prevented CaSR expression (western blot), whereas the G774S mutation determined a reduced receptor sensitivity to calcium (IP3 assay). PHPT patients showed significantly (P < 0.001) higher serum calcium, parathyroid hormone, urinary calcium and calcium-creatinine clearance ratio (CCCR) and significantly lower serum phosphate than FHH ones.
  
  
  CONCLUSIONS
  FHH should be clearly differentiated by PHPT to avoid unnecessary surgery: CCCR could be a useful screening tool while genetic analysis should include the two novel CaSR mutations herein described. The role of multiple polymorphisms deserves further investigation in patients with an FHH phenotype.
authors.affiliation: Department of Translational Medicine, Nephrology and Transplantation, University 'Amedeo Avogadro', Hospital 'Maggiore della Carità', Novara, NO, Italy.
authors.forename: Piero
authors.initials: P
authors.lastname: Stratta
authors.affiliation: Department of Translational Medicine, Nephrology and Transplantation, University 'Amedeo Avogadro', Hospital 'Maggiore della Carità', Novara, NO, Italy.
authors.forename: Guido
authors.initials: G
authors.lastname: Merlotti
authors.affiliation: Department of Translational Medicine, Nephrology and Transplantation, University 'Amedeo Avogadro', Hospital 'Maggiore della Carità', Novara, NO, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Musetti
authors.affiliation: Department of Translational Medicine, Nephrology and Transplantation, University 'Amedeo Avogadro', Hospital 'Maggiore della Carità', Novara, NO, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Quaglia
authors.affiliation: Department of Health Sciences, SCDU of Pediatrics, University 'Amedeo Avogadro', Hospital 'Maggiore della Carità',  Novara, NO, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Pagani
authors.affiliation: Department of Translational Medicine, Nephrology and Transplantation, University 'Amedeo Avogadro', Hospital 'Maggiore della Carità', Novara, NO, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Izzo
authors.affiliation: Department of Translational Medicine, Nephrology and Transplantation, University 'Amedeo Avogadro', Hospital 'Maggiore della Carità', Novara, NO, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Radin
authors.affiliation: Department of Translational Medicine, Nephrology and Transplantation, University 'Amedeo Avogadro', Hospital 'Maggiore della Carità', Novara, NO, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Airoldi
authors.affiliation: Medical Genetics, IRCCS 'Casa Sollievo della Sofferenza' Hospital, San Giovanni Rotondo, FG, Italy.
authors.forename: Filomena
authors.initials: F
authors.lastname: Baorda
authors.affiliation: Medical Genetics, IRCCS 'Casa Sollievo della Sofferenza' Hospital, San Giovanni Rotondo, FG, Italy.
authors.forename: Teresa
authors.initials: T
authors.lastname: Palladino
authors.affiliation: Medical Genetics, IRCCS 'Casa Sollievo della Sofferenza' Hospital, San Giovanni Rotondo, FG, Italy.
authors.forename: Maria Pia
authors.initials: MP
authors.lastname: Leone
authors.affiliation: Medical Genetics, IRCCS 'Casa Sollievo della Sofferenza' Hospital, San Giovanni Rotondo, FG, Italy.
authors.forename: Vito
authors.initials: V
authors.lastname: Guarnieri
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000235:genetics; D034002:Adaptor Protein Complex sigma Subunits / Q000235:genetics; D000328:Adult; D000368:Aged; D015153:Blotting, Western; D015331:Cohort Studies; D004247:DNA / Q000235:genetics; D003937:Diagnosis, Differential; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics; D006801:Humans; D006934:Hypercalcemia / Q000151:congenital* / Q000175:diagnosis / Q000235:genetics; D049950:Hyperparathyroidism, Primary / Q000175:diagnosis / Q000235:genetics*; D007558:Italy / Q000453:epidemiology; D008297:Male; D008875:Middle Aged; D009154:Mutation / Q000235:genetics*; D010281:Parathyroid Hormone / Q000097:blood; D011110:Polymorphism, Genetic / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D044169:Receptors, Calcium-Sensing / Q000235:genetics* / Q000378:metabolism
chemical_list: C510065:AP2S1 protein, human; D033962:Adaptor Protein Complex 2; D034002:Adaptor Protein Complex sigma Subunits; C095550:CASR protein, human; C554062:GNA11 protein, human; D044385:GTP-Binding Protein alpha Subunits; D010281:Parathyroid Hormone; D044169:Receptors, Calcium-Sensing; D004247:DNA
keywords: AP2S1; CaSR; GNA11; familial hypocalciuric hypercalcemia; hypercalcaemia