title: Molecular and clinical analysis of a neonatal severe hyperparathyroidism case caused by a stop mutation in the calcium-sensing receptor extracellular domain representing in effect a human 'knockout'.
abstract: OBJECTIVE
  Loss-of-function calcium-sensing receptor (CAR) mutations cause elevated parathyroid hormone (PTH) secretion and hypercalcaemia. Although full Car deletion is possible in mice, most human CAR mutations result from a single amino acid substitution that maintains partial function. However, here, we report a case of neonatal severe hyperparathyroidism (NSHPT) in which the truncated CaR lacks any transmembrane domain (CaR(R392X)), in effect a full CAR 'knockout'.
  
  
  METHODS
  The infant (daughter of distant cousins) presented with hypercalcaemia (5.5-6  mmol/l corrected calcium (2.15-2.65)) and elevated PTH concentrations (650-950  pmol/l (12-81)) together with skeletal demineralisation. NSHPT was confirmed by CAR gene sequencing (homozygous c.1174C-to-T mutation) requiring total parathyroidectomy during which only two glands were located and removed, resulting in normalisation of her serum PTH/calcium levels.
  
  
  METHODS
  The R392X stop codon was inserted into human CAR and the resulting mutant (CaR(R392X)) expressed transiently in HEK-293 cells.
  
  
  RESULTS
  CaR(R392X) expressed as a 54  kDa dimeric glycoprotein that was undetectable in conditioned medium or in the patient's urine. The membrane localisation observed for wild-type CaR in parathyroid gland and transfected HEK-293 cells was absent from the proband's parathyroid gland and from CaR(R392X)-transfected cells. Expression of the mutant was localised to endoplasmic reticulum consistent with its lack of functional activity.
  
  
  CONCLUSIONS
  Intriguingly, the patient remained normocalcaemic throughout childhood (2.5 mM corrected calcium, 11 pg/ml PTH (10-71), age 8 years) but exhibited mild asymptomatic hypocalcaemia at age 10 years, now treated with 1-hydroxycholecalciferol and Ca2+ supplementation. Despite representing a virtual CAR knockout, the patient displays no obvious pathologies beyond her calcium homeostatic dysfunction.
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK. d.ward@manchester.ac.uk
authors.forename: D T
authors.initials: DT
authors.lastname: Ward
authors.forename: M Z
authors.initials: MZ
authors.lastname: Mughal
authors.forename: M
authors.initials: M
authors.lastname: Ranieri
authors.forename: M M
authors.initials: MM
authors.lastname: Dvorak-Ewell
authors.forename: G
authors.initials: G
authors.lastname: Valenti
authors.forename: D
authors.initials: D
authors.lastname: Riccardi
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine; D002118:Calcium / Q000097:blood; D002648:Child; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D057809:HEK293 Cells; D006801:Humans; D006934:Hypercalcemia / Q000097:blood / Q000209:etiology*; D006961:Hyperparathyroidism / Q000097:blood / Q000151:congenital / Q000175:diagnosis* / Q000235:genetics*; D015151:Immunoblotting; D007223:Infant; D007231:Infant, Newborn; D016254:Mutagenesis, Insertional; D010281:Parathyroid Hormone / Q000097:blood / Q000235:genetics; D016105:Parathyroidectomy / Q000379:methods; D044169:Receptors, Calcium-Sensing / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods; D012720:Severity of Illness Index; D014162:Transfection; D016896:Treatment Outcome
chemical_list: D010281:Parathyroid Hormone; D044169:Receptors, Calcium-Sensing; D001120:Arginine; D002118:Calcium