Autosomal dominant hypocalcemia in monozygotic twins caused by a de novo germline mutation near the amino-terminus of the human calcium receptor

Jianxin Hu, Stefano Mora, Giovanna Weber, Ilaria Zamproni, Maria Carla Proverbio, Allen M. Spiegel

Research output: Contribution to journalArticle

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Abstract

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. Introduction: Activating mutations of the Ca2+-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. Materials and 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.

Original languageEnglish (US)
Pages (from-to)578-586
Number of pages9
JournalJournal of Bone and Mineral Research
Volume19
Issue number4
DOIs
StatePublished - Apr 2004
Externally publishedYes

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Calcium-Sensing Receptors
Monozygotic Twins
Germ-Line Mutation
Siblings
Hypocalcemia
Mutation
Missense Mutation
Droseraceae
Molecular Biology
Parents
Familial Hypercalciuric Hypocalcemia
HEK293 Cells
Phosphatidylinositols
Aspartic Acid
Mutagenesis
Genes
Arginine
Hydrolysis
Complementary DNA
Salts

Keywords

  • Activating mutation
  • Autosomal dominant hypocalcemia
  • Human calcium receptor
  • Loop 2
  • Venus flytrap-like domain

ASJC Scopus subject areas

  • Surgery

Cite this

Autosomal dominant hypocalcemia in monozygotic twins caused by a de novo germline mutation near the amino-terminus of the human calcium receptor. / Hu, Jianxin; Mora, Stefano; Weber, Giovanna; Zamproni, Ilaria; Proverbio, Maria Carla; Spiegel, Allen M.

In: Journal of Bone and Mineral Research, Vol. 19, No. 4, 04.2004, p. 578-586.

Research output: Contribution to journalArticle

Hu, Jianxin ; Mora, Stefano ; Weber, Giovanna ; Zamproni, Ilaria ; Proverbio, Maria Carla ; Spiegel, Allen M. / Autosomal dominant hypocalcemia in monozygotic twins caused by a de novo germline mutation near the amino-terminus of the human calcium receptor. In: Journal of Bone and Mineral Research. 2004 ; Vol. 19, No. 4. pp. 578-586.
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abstract = "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. Introduction: Activating mutations of the Ca2+-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. Materials and 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.",
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AU - Proverbio, Maria Carla

AU - Spiegel, Allen M.

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