title: Identification and functional characterization of novel calcium-sensing receptor mutations in familial hypocalciuric hypercalcemia and autosomal dominant hypocalcemia.
abstract: Familial hypocalciuric hypercalcemia (FHH), neonatal severe hyperparathyroidism (NSHPT), and autosomal dominant hypocalcemia (ADH), in which calcium homeostasis is disordered, are associated with mutations in the calcium-sensing receptor (CASR). Six unrelated kindreds with FHH and/or NSHPT and two unrelated kindreds with ADH were studied. Direct sequence analysis of the exons of the CASR gene identified heterozygous mutations in six of the kindreds with FHH and in one of those with ADH. We performed functional analyses on the novel missense and insertion/frameshift mutants by transiently transfecting wild-type and mutant CASRs tagged with a c-Myc epitope in human embryonic kidney (HEK293) cells. All mutant receptors were expressed at a similar level to that of the wild type; however, whereas mutants R220W and A835T (the ADH mutant) were fully glycosylated and were visualized on the cell surface, glycosylation of mutants G549R and C850-851 ins/fs was impaired, resulting in reduced cell surface staining. In fura-2-loaded HEK293 cells expressing the wild-type or mutant receptors, the inactivating R220W mutant produced a significant shift to the right relative to the wild-type CASR in the cytosolic calcium response to increasing extracellular calcium concentrations and the G549R and C850-851 ins/fs mutants were without detectable activity. The activating A835T mutation resulted in a shift to the left in the cytosolic calcium response to extracellular calcium concentrations relative to the wild type. Our studies have identified novel CASR mutations that alter the function of the CASR in several different ways.
authors.affiliation: Department of Medicine, McGill University and Calcium Research Laboratory, Royal Victoria Hospital, Montréal, Quebéc, Canada H3A 1A1.
authors.forename: Lilia
authors.initials: L
authors.lastname: D'Souza-Li
authors.forename: Bing
authors.initials: B
authors.lastname: Yang
authors.forename: Lucie
authors.initials: L
authors.lastname: Canaff
authors.forename: Mei
authors.initials: M
authors.lastname: Bai
authors.forename: David A
authors.initials: DA
authors.lastname: Hanley
authors.forename: Murat
authors.initials: M
authors.lastname: Bastepe
authors.forename: Sonia R
authors.initials: SR
authors.lastname: Salisbury
authors.forename: Edward M
authors.initials: EM
authors.lastname: Brown
authors.forename: David E C
authors.initials: DE
authors.lastname: Cole
authors.forename: Geoffrey N
authors.initials: GN
authors.lastname: Hendy
mesh_terms: D002118:Calcium / Q000378:metabolism / Q000652:urine*; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005799:Genes, Dominant; D006031:Glycosylation; D006801:Humans; D006934:Hypercalcemia / Q000235:genetics* / Q000652:urine*; D006961:Hyperparathyroidism / Q000235:genetics / Q000378:metabolism; D007231:Infant, Newborn; D007425:Intracellular Membranes / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation / Q000502:physiology*; D009994:Osmolar Concentration; D010375:Pedigree; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D012016:Reference Values
chemical_list: D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D002118:Calcium