Knock-in Mouse with Mutant Gα11: Characterization of Phenotype and Pharmacological Rescue of Hypocalcemia By Inhibitors

Presentation Number: SAT 356
Date of Presentation: April 1st, 2017

Kelly Lauter Roszko*1, Ruiye Bi1, Caroline M Gorvin2, Hans brauner-Osborne3, Xiao-Feng Xiong4, Asuka Inoue5, Rajesh V Thakker2, Kristian Strømgaard4, Thomas J Gardella1 and Michael Mannstadt1
1Massachusetts General Hospital and Harvard Medical School, Boston, MA, 2University of Oxford, Oxford, United Kingdom, 3University of Copenhagen, Copenhagan, Denmark, 4University of Copenhagen, Copenhagen, Denmark, 5Tohoku University, Japan

Abstract

Parathyroid hormone (PTH) acts to maintain extracellular calcium in a tight range. Low PTH levels result in hypocalcemia and hyperphosphatemia. Autosomal dominant hypocalcemia type 2 (ADH2) is caused by activating mutations in GNA11, encoding Gα11, which activate the CASR pathway. One such mutation, found in a family with ADH2, is the heterozygous missense mutation c.178C->T leading to the replacement of arginine 60 with cysteine in helix α1 of the GTPase domain.

We expressed wildtype (WT) Arg60 and mutant Cys60 Gα11 in HEK293 cells stably expressing the CASR and measured the intracellular calcium response to changes in extracellular calcium concentrations. The leftward shift of the concentration-response curve in the cells expressing mutant Gα11 illustrated an increased sensitivity to calcium. We treated these cells with the calcilytic NPS 2143 and the EC50of the mutant cells returned to that of WT cells.

With CRISPR/Cas9 technology, we created a mouse model harboring the Arg60Cys mutation. Compared to WT littermates, nine-week-old mice heterozygous and homozygous for Gα11 R60C were hypocalcemic. Compared to WT animals, PTH was inappropriate in heterozygous and homozygous animals. Serum phosphate was higher in homozygous mice. We also noted increased pigmentation of the tails and ears of mutant mice, implicating the activation of other GPCRs by the mutant Gα11. μCT analysis of 12-week old mice revealed lower bone mineral density (BMD) and bone volume/total volume (BV/TV) of female heterozygous/homozygous and male heterozygous mice. This finding was surprising given that humans with hypoparathyroidism typically have increased BMD. The urinary fractional excretion index of calcium (FECa) was not different in mutant mice, raising the possibility that Gα11 does not have a dominant effect on CASR function in the distal tubule cells.

We injected 30 mg/kg of NPS 2143 i.p.; 4 hours after injection, serum calcium was increased in mice WT, heterozygous and homozygous for R60C. Serum PTH increased in all animal groups, and serum phosphate increased in WT mice. The urinary FECa decreased in heterozygous and homozygous animals. We tested the effects of the specific Gα11/Gαq inhibitor YM-254890. A single dose of 0.15mg/kg YM-254890 i.p. increased the calcium in WT and heterozygous R60C mice. Published crystallography studies predict that YM-254890 inhibits WT Gα11, but not Gα11 R60C, consistent with our findings of no change in serum calcium in response to treatment of R60C homozygous animals.

We have used CRISPR/Cas9 to create a mouse with the p.Arg60Cys mutation found in humans with ADH2. Our mouse model mimics the biochemical findings of the human disease, i.e. hypocalcemia and inadequate PTH. Mutant mice also have decreased BMD, increased pigmentation, and unchanged urinary FECa. This mouse model has identified phenotypes, in addition to hypocalcemia, associated with Gα11 mutations.

 

Disclosure: KS: Founder, Avilex Pharma. MM: Advisory Group Member, NPS-Shire. Nothing to Disclose: KL, RB, CMG, HB, XFX, AI, RVT, TJG