Dataset Information

Mutational analysis of the adaptor protein 2 sigma subunit (AP2S1) gene: search for autosomal dominant hypocalcemia type 3 (ADH3).

ABSTRACT: Autosomal dominant hypocalcemia (ADH) types 1 and 2 are due to calcium-sensing receptor (CASR) and G-protein subunit-?11 (GNA11) gain-of-function mutations, respectively, whereas CASR and GNA11 loss-of-function mutations result in familial hypocalciuric hypercalcemia (FHH) types 1 and 2, respectively. Loss-of-function mutations of adaptor protein-2 sigma subunit (AP2? 2), encoded by AP2S1, cause FHH3, and we therefore sought for gain-of-function AP2S1 mutations that may cause an additional form of ADH, which we designated ADH3.The objective of the study was to investigate the hypothesis that gain-of-function AP2S1 mutations may cause ADH3.The sample size required for the detection of at least one mutation with a greater than 95% likelihood was determined by binomial probability analysis. Nineteen patients (including six familial cases) with hypocalcemia in association with low or normal serum PTH concentrations, consistent with ADH, but who did not have CASR or GNA11 mutations, were ascertained. Leukocyte DNA was used for sequence and copy number variation analysis of AP2S1.Binomial probability analysis, using the assumption that AP2S1 mutations would occur in hypocalcemic patients at a prevalence of 20%, which is observed in FHH patients without CASR or GNA11 mutations, indicated that the likelihood of detecting at least one AP2S1 mutation was greater than 95% and greater than 98% in sample sizes of 14 and 19 hypocalcemic patients, respectively. AP2S1 mutations and copy number variations were not detected in the 19 hypocalcemic patients.The absence of AP2S1 abnormalities in hypocalcemic patients, suggests that ADH3 may not occur or otherwise represents a rare hypocalcemic disorder.

SUBMITTER: Rogers A

PROVIDER: S-EPMC4447854 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Mutational analysis of the adaptor protein 2 sigma subunit (AP2S1) gene: search for autosomal dominant hypocalcemia type 3 (ADH3).

Rogers Angela A Nesbit M Andrew MA Hannan Fadil M FM Howles Sarah A SA Gorvin Caroline M CM Cranston Treena T Allgrove Jeremy J Bevan John S JS Bano Gul G Brain Caroline C Datta Vipan V Grossman Ashley B AB Hodgson Shirley V SV Izatt Louise L Millar-Jones Lynne L Pearce Simon H SH Robertson Lisa L Selby Peter L PL Shine Brian B Snape Katie K Warner Justin J Thakker Rajesh V RV

The Journal of clinical endocrinology and metabolism 20140407 7

<h4>Context</h4>Autosomal dominant hypocalcemia (ADH) types 1 and 2 are due to calcium-sensing receptor (CASR) and G-protein subunit-α11 (GNA11) gain-of-function mutations, respectively, whereas CASR and GNA11 loss-of-function mutations result in familial hypocalciuric hypercalcemia (FHH) types 1 and 2, respectively. Loss-of-function mutations of adaptor protein-2 sigma subunit (AP2σ 2), encoded by AP2S1, cause FHH3, and we therefore sought for gain-of-function AP2S1 mutations that may cause an ...[more]

PMID: 24708097

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Project description:The adaptor protein-2 sigma subunit (AP2σ), encoded by AP2S1, forms a heterotetrameric complex, with AP2α, AP2β, and AP2μ subunits, that is pivotal for clathrin-mediated endocytosis, and AP2σ loss-of-function mutations impair internalization of the calcium-sensing receptor (CaSR), a G-protein-coupled receptor, and cause familial hypocalciuric hypercalcemia type-3 (FHH3). Mice with AP2σ mutations that would facilitate investigations of the in vivo role of AP2σ, are not available, and we therefore embarked on establishing such mice. We screened >10,000 mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) for Ap2s1 mutations and identified 5 Ap2s1 variants, comprising 2 missense (Tyr20Asn and Ile123Asn) and 3 intronic base substitutions, one of which altered the invariant donor splice site dinucleotide gt to gc. Three-dimensional modeling and cellular expression of the missense Ap2s1 variants did not reveal them to alter AP2σ structure or CaSR-mediated signaling, but investigation of the donor splice site variant revealed it to result in an in-frame deletion of 17 evolutionarily conserved amino acids (del17) that formed part of the AP2σ α1-helix, α1-β3 loop, and β3 strand. Heterozygous mutant mice (Ap2s1+/del17 ) were therefore established, and these had AP2σ haplosufficiency but were viable with normal appearance and growth. Ap2s1+/del17 mice, when compared with Ap2s1+/+ mice, also had normal plasma concentrations of calcium, phosphate, magnesium, creatinine, urea, sodium, potassium, and alkaline phosphatase activity; normal urinary fractional excretion of calcium, phosphate, sodium, and potassium; and normal plasma parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (1,25(OH)2) concentrations. However, homozygous Ap2s1del17/del17 mice were non-viable and died between embryonic days 3.5 and 9.5 (E3.5-9.5), thereby indicating that AP2σ likely has important roles at the embryonic patterning stages and organogenesis of the heart, thyroid, liver, gut, lungs, pancreas, and neural systems. Thus, our studies have established a mutant mouse model that is haplosufficient for AP2σ.

Project description:Autosomal dominant hypocalcemia type 1 (ADH1) is a rare form of hypoparathyroidism caused by heterozygous, gain-of-function mutations of the calcium-sensing receptor gene (CAR). Individuals are hypocalcemic with inappropriately low parathyroid hormone (PTH) secretion and relative hypercalciuria. Calcilytics are negative allosteric modulators of the extracellular calcium receptor (CaR) and therefore may have therapeutic benefits in ADH1. Five adults with ADH1 due to four distinct CAR mutations received escalating doses of the calcilytic compound NPSP795 (SHP635) on 3 consecutive days. Pharmacokinetics, pharmacodynamics, efficacy, and safety were assessed. Parallel in vitro testing with subject CaR mutations assessed the effects of NPSP795 on cytoplasmic calcium concentrations (Ca2+ i ), and ERK and p38MAPK phosphorylation. These effects were correlated with clinical responses to administration of NPSP795. NPSP795 increased plasma PTH levels in a concentration-dependent manner up to 129% above baseline (p = 0.013) at the highest exposure levels. Fractional excretion of calcium (FECa) trended down but not significantly so. Blood ionized calcium levels remained stable during NPSP795 infusion despite fasting, no calcitriol supplementation, and little calcium supplementation. NPSP795 was generally safe and well-tolerated. There was significant variability in response clinically across genotypes. In vitro, all mutant CaRs were half-maximally activated (EC50 ) at lower concentrations of extracellular calcium (Ca2+ o ) compared to wild-type (WT) CaR; NPSP795 exposure increased the EC50 for all CaR activity readouts. However, the in vitro responses to NPSP795 did not correlate with any clinical parameters. NPSP795 increased plasma PTH levels in subjects with ADH1 in a dose-dependent manner, and thus, serves as proof-of-concept that calcilytics could be an effective treatment for ADH1. Albeit all mutations appear to be activating at the CaR, in vitro observations were not predictive of the in vivo phenotype or the response to calcilytics, suggesting that other parameters impact the response to the drug. © 2019 American Society for Bone and Mineral Research.

Project description:Autosomal dominant hypocalcemia (ADH) is characterized by hypocalcemia, inappropriately low serum parathyroid hormone concentrations and hypercalciuria. ADH is genetically heterogeneous with ADH type 1 (ADH1), the predominant form, being caused by germline gain-of-function mutations of the G-protein coupled calcium-sensing receptor (CaSR), and ADH2 caused by germline gain-of-function mutations of G-protein subunit α-11 (Gα11 ). To date Gα11 mutations causing ADH2 have been reported in only five probands. We investigated a multigenerational nonconsanguineous family, from Iran, with ADH and keratoconus which are not known to be associated, for causative mutations by whole-exome sequencing in two individuals with hypoparathyroidism, of whom one also had keratoconus, followed by cosegregation analysis of variants. This identified a novel heterozygous germline Val340Met Gα11 mutation in both individuals, and this was also present in the other two relatives with hypocalcemia that were tested. Three-dimensional modeling revealed the Val340Met mutation to likely alter the conformation of the C-terminal α5 helix, which may affect G-protein coupled receptor binding and G-protein activation. In vitro functional expression of wild-type (Val340) and mutant (Met340) Gα11 proteins in HEK293 cells stably expressing the CaSR, demonstrated that the intracellular calcium responses following stimulation with extracellular calcium, of the mutant Met340 Gα11 led to a leftward shift of the concentration-response curve with a significantly (p < 0.0001) reduced mean half-maximal concentration (EC50 ) value of 2.44 mM (95% CI, 2.31 to 2.77 mM) when compared to the wild-type EC50 of 3.14 mM (95% CI, 3.03 to 3.26 mM), consistent with a gain-of-function mutation. A novel His403Gln variant in transforming growth factor, beta-induced (TGFBI), that may be causing keratoconus was also identified, indicating likely digenic inheritance of keratoconus and ADH2 in this family. In conclusion, our identification of a novel germline gain-of-function Gα11 mutation, Val340Met, causing ADH2 demonstrates the importance of the Gα11 C-terminal region for G-protein function and CaSR signal transduction. © 2016 American Society for Bone and Mineral Research.

Project description:Calcilytics are calcium-sensing receptor (CaSR) antagonists that reduce the sensitivity of the CaSR to extracellular calcium. Calcilytics have the potential to treat autosomal dominant hypocalcemia type 1 (ADH1), which is caused by germline gain-of-function CaSR mutations and leads to symptomatic hypocalcemia, inappropriately low PTH concentrations, and hypercalciuria. To date, only one calcilytic compound, NPSP795, has been evaluated in patients with ADH1: Doses of up to 30 mg per patient have been shown to increase PTH concentrations, but did not significantly alter ionized blood calcium concentrations. The aim of this study was to further investigate NPSP795 for the treatment of ADH1 by undertaking in vitro and in vivo studies involving Nuf mice, which have hypocalcemia in association with a gain-of-function CaSR mutation, Leu723Gln. Treatment of HEK293 cells stably expressing the mutant Nuf (Gln723) CaSR with 20nM NPSP795 decreased extracellular Ca2+-mediated intracellular calcium and phosphorylated ERK responses. An in vivo dose-ranging study was undertaken by administering a s.c. bolus of NPSP795 at doses ranging from 0 to 30 mg/kg to heterozygous (Casr +/Nuf ) and to homozygous (Casr Nuf/Nuf ) mice, and measuring plasma PTH responses at 30 min postdose. NPSP795 significantly increased plasma PTH concentrations in a dose-dependent manner with the 30 mg/kg dose causing a maximal (≥10-fold) rise in PTH. To determine whether NPSP795 can rectify the hypocalcemia of Casr +/Nuf and Casr Nuf/Nuf mice, a submaximal dose (25 mg/kg) was administered, and plasma adjusted-calcium concentrations measured over a 6-hour period. NPSP795 significantly increased plasma adjusted-calcium in Casr +/Nuf mice from 1.87 ± 0.03 mmol/L to 2.16 ± 0.06 mmol/L, and in Casr Nuf/Nuf mice from 1.70 ± 0.03 mmol/L to 1.89 ± 0.05 mmol/L. Our findings show that NPSP795 elicits dose-dependent increases in PTH and ameliorates the hypocalcemia in an ADH1 mouse model. Thus, calcilytics such as NPSP795 represent a potential targeted therapy for ADH1. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Dataset Information

Mutational analysis of the adaptor protein 2 sigma subunit (AP2S1) gene: search for autosomal dominant hypocalcemia type 3 (ADH3).

Publications

Mutational analysis of the adaptor protein 2 sigma subunit (AP2S1) gene: search for autosomal dominant hypocalcemia type 3 (ADH3).

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