Autosomal dominant hypocalcemia in monozygotic twins caused by a de novo germline mutation near the amino-terminus of the human calcium receptor.
To define the molecular pathogenesis of severe postnatal hypocalcemia in monozygotic twin sisters, we sequenced their CaR gene and identified a missense mutation, K29E. Expression of the mutant receptor in vitro showed a marked increase in Ca2+ sensitivity explaining the observed phenotype. Additional mutagenesis studies lead us to speculate concerning a novel mechanism whereby the K29E mutation may lead to receptor activation.


BACKGROUND
Activating mutations of the Ca(2+)-sensing receptor (CaR) gene have been identified in subjects with autosomal dominant hypocalcemia. Study of such mutations has provided insight into the mechanism of activation of the CaR.


METHODS
We performed biochemical and molecular genetic studies on monozygotic twin sisters who presented with early postnatal hypocalcemia and on their unaffected sister and parents. Functional characterization of mutant CaRs transfected in HEK-293 cells included immunoblots to monitor protein expression and Ca2+ stimulation of phosphoinositide hydrolysis to measure Ca2+ sensitivity.


RESULTS
We identified a K29E missense mutation in the twin sisters but not in their parents or unaffected sister. The K29E mutant CaR showed a marked increase in Ca2+ sensitivity, including when it was co-transfected with wildtype CaR cDNA, consistent with a dominant effect. Substitution of K29 by aspartate equivalently increased CaR sensitivity, whereas conservative substitution by arginine did not.


CONCLUSIONS
Severe postnatal hypocalcemia in the twin sisters was caused by a de novo germline activating mutation. In a model of the Venus flytrap-like domain of the extracellular amino-terminus of the CaR, K29 is located close to a peptide loop, "loop 2," that forms part of the dimer interface and is the site of 10 of the previously reported naturally occurring activating CaR mutations. We speculate that K29E increases Ca2+ sensitivity of the CaR by disrupting a salt bridge between K29 and an acidic residue in loop 2 and thereby changes the normal structure of loop 2 that maintains the CaR in its inactive conformation.
Molecular Pathophysiology Section, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892, USA. jianxinh@intra.niddk.nih.gov
Jianxin
J
Hu
Stefano
S
Mora
Giovanna
G
Weber
Ilaria
I
Zamproni
Maria Carla
MC
Proverbio
Allen M
AM
Spiegel
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002118:Calcium / Q000097:blood*; D002460:Cell Line; D005260:Female; D015870:Gene Expression; D005799:Genes, Dominant; D018095:Germ-Line Mutation; D006801:Humans; D006996:Hypocalcemia / Q000097:blood / Q000151:congenital / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010375:Pedigree; D017434:Protein Structure, Tertiary / Q000235:genetics; D044169:Receptors, Calcium-Sensing / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014430:Twins, Monozygotic / Q000235:genetics*
D044169:Receptors, Calcium-Sensing; D002118:Calcium