title: Two Italian kindreds with familial hypocalciuric hypercalcaemia caused by loss-of-function mutations in the calcium-sensing receptor (CaR) gene: functional characterization of a novel CaR missense mutation.
abstract: OBJECTIVE
  Description of two unrelated Italian kindreds with familial hypocalciuric hypercalcaemia (FHH), an autosomal dominant disease mostly caused by heterozygous inactivating mutations of the Ca2+ sensing receptor (CaR).
  
  
  METHODS
  We studied 11 members of the two families. Genomic DNA was isolated from peripheral blood leucocytes in all family members and in 50 unrelated Italian controls. Total serum and ionized calcium, PTH, creatinine, phosphate, magnesium, and urinary calcium clearance to creatinine clearance ratio were measured. Direct sequencing of the entire coding region of the CaR was performed in the probands. Functional studies were performed in COS-7 cells transiently expressing the mutated CaR.
  
  
  RESULTS
  In the proband of family A direct sequencing revealed a novel heterozygous Y218C missense mutation in exon 4. The same mutation was identified in the affected but not in the unaffected family members or in any of the 50 unrelated Italian controls. Transient expression of the Y218C CaR in COS-7 cells revealed a blunted Ca2+-evoked accumulation of inositol trisphosphates, indicating that the Y218C is a loss-of-function mutation. Cotransfection experiments showed that the mutant receptor had no impact on the function of the wild-type receptor, suggesting that a reduced expression of the normal CaR, rather than a dominant-negative effect, accounted for the functional impairment. In the proband of family B an already described heterozygous P55L missense mutation in exon 2 of the CaR gene was found. The same mutation was identified in the affected family members.
  
  
  CONCLUSIONS
  We described two familial hypocalciuric hypercalcaemia kindreds with loss-of-function mutations of the Ca2+ receptor gene and identified a novel heterozygous mutation (Y218C) characterized by a blunted response to Ca2+ stimulation compared to the wild-type receptor and no interference with the function of the wild-type Ca2+ receptor.
authors.affiliation: Dipartimento di Endocrinologia e Metabolismo, Università di Pisa, Pisa, Italy.
authors.forename: Filomena
authors.initials: F
authors.lastname: Cetani
authors.forename: Elena
authors.initials: E
authors.lastname: Pardi
authors.forename: Simona
authors.initials: S
authors.lastname: Borsari
authors.forename: Massimo
authors.initials: M
authors.lastname: Tonacchera
authors.forename: Eugenia
authors.initials: E
authors.lastname: Morabito
authors.forename: Aldo
authors.initials: A
authors.lastname: Pinchera
authors.forename: Claudio
authors.initials: C
authors.lastname: Marcocci
authors.forename: Glada
authors.initials: G
authors.lastname: Dipollina
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D019556:COS Cells / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology / Q000652:urine*; D005260:Female; D006111:Graves Disease / Q000235:genetics; D006801:Humans; D006934:Hypercalcemia / Q000235:genetics*; D007295:Inositol Phosphates / Q000378:metabolism; D007558:Italy; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D010375:Pedigree; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014162:Transfection
chemical_list: D007295:Inositol Phosphates; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D002118:Calcium